Difference between revisions of "Team:Stanford-Brown/SB16 Notebooks p-Aramid"

 
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            <li><a href="https://2016.igem.org/Team:Stanford-Brown">Home</a></li>
 
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                    <li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioMembrane_Latex">Latex</a></li>
 
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                    <li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Float_Overview">Overview</a></li>
 
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Float_Gas">Gas production</a></li>
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Float_Gas">Gas production</a></li>
 
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_Chromoproteins">Chromoproteins</a></li>
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_Chromoproteins">Chromoproteins</a></li>
 
                    <li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_FQsensor">Fluorophore-Quencher</a></li>
 
                    <li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_FQsensor">Fluorophore-Quencher</a></li>
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_BioMembrane_AptamerPurification">Aptamer purification</a></li>
 
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/Integrated_Practices">Integrated Human Practices</a></li>
 
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_Chromoproteins">Chromoproteins</a></li>
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_Chromoproteins">Chromoproteins</a></li>
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_FQsensor">Fluorophore-Quencher</a></li>
 
<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_FQsensor">Fluorophore-Quencher</a></li>
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_Nylon">Nylon</a></li>
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<li><a href="https://2016.igem.org/Team:Stanford-Brown/SB16_Notebooks_Interlab">Interlab Study</a></li>
 
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<title>pABA future · Benchling</title>
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<body><div><div class="printable-container"><div><div class="printable-page-container"><div class="noteEditor mediocre" tabindex="-1"><div></div><div class="noteEditor-container"><div class="u-cursor-text noteEditor-catcher u-disable-margin-collapse dnd-drop-container"><div class="noteEditor-contentWrapper"><h1 class="mediocre-titleEditor"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="notetitleeditor" data-index="0">pABA future</div></h1><div><div><i class="faded">Made with Benchling</i></div><div><b>Project:</b> iGEM 2016</div><div><b>Authors:</b> Eric Liu, Anna Le</div><div><b>Date:</b> 2016-08-30</div></div><div class="editor" data-editor="notes"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="daySeparator"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 8/30</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3649" data-index="0" style="margin-left:0;">**First few steps of each project are definitely doable and prob wont take too much time!** Currently there are 3 subprojects under pABA. In the end we hope to consolidate these subprojects into one. Our subprojects are:</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>1.</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3650" data-index="1" style="margin-left:35px;">Introducing extra copies of pab,A,B,C to increase pABA production</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>2.</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3651" data-index="2" style="margin-left:35px;">Knocking out the last enzyme of the pathway to stop pABA from being further converted into folate</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>3.</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3652" data-index="3" style="margin-left:35px;">Overproducing the first enzyme in the pathway</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3653" data-index="4" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3654" data-index="5" style="margin-left:0;"><b>Inventory</b></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3655" data-index="6" style="margin-left:35px;">ELIM DNA tubes marked with blue X's along bottom row = don't use</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3656" data-index="7" style="margin-left:35px;">Pink 1.5 mL tubes with "25" on them = miniprepped pab(A+B) cultures</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3657" data-index="8" style="margin-left:35px;">WHITE "Psb1c3 bb __ PCR 9/1" tubesand BLUE "9/2 1/2/1b/2b" tubes are trash lol.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3658" data-index="9" style="margin-left:35px;">"pcas9 478" tube = undigested pcas9 plasmid that needs to be digested for Project #2. Plasmid size is 10k</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3659" data-index="10" style="margin-left:35px;">Primers</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3660" data-index="11" style="margin-left:70px;">"psb1c3 backbone"</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3661" data-index="12" style="margin-left:70px;">"c gibson" = primers for pabC to make gibson ends</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3662" data-index="13" style="margin-left:70px;">"dahp" - primers for DAHP synthase gene to make gibson ends</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3663" data-index="14" style="margin-left:35px;"><span style="color:#344043"><span style="background-color:#FFFFFF">pc9 PCR tube is probably useless.</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3664" data-index="15" style="margin-left:35px;">General sizes</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3665" data-index="16" style="margin-left:70px;">pcas9 = 10k</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3666" data-index="17" style="margin-left:70px;">psb1c3 backbone (const. promoter + terminator) = 2.7kb</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3667" data-index="18" style="margin-left:70px;">blitzen blue backbone = 4kb</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span>○</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3668" data-index="19" style="margin-left:70px;">dahp synthase = 1k</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3669" data-index="20" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3670" data-index="21" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-header1"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3671" data-index="22" style="margin-left:0;"><u>To do</u></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-header2"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3672" data-index="23" style="margin-left:0;">Project 1: Extra copies of pabA,B,C to increase pABA production</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3673" data-index="24" style="margin-left:35px;">Biobrick pabC</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3674" data-index="25" style="margin-left:70px;"><b>DAY 1 </b>Transform PabA+B (DNA is in iGEM 2015 distribution kit #2) under 13P into NEB 5alpha cells. (Not making protein, so no need to use T7 cells. Also 5-alpha is more efficient anyways.) --- <b><span style="color:#84C620">AL - I've done this already, I'm leaving the plates out over the weekend (9/3-9/5). They should be by the RT shaker)</span></b></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3675" data-index="26" style="margin-left:105px;">Amy redid this 9/6 because 9/2 transformation failed.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span>■</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3676" data-index="27" style="margin-left:105px;">Redid transformation 9/14.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:140px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3677" data-index="28" style="margin-left:140px;">Sequenced colonies, ran gels - gels did not have bands 2.7kb!! And sequencing results were trash as well!!</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3678" data-index="29" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3679" data-index="30" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3680" data-index="31" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3681" data-index="32" style="margin-left:70px;"><b>DAY 2 </b>If transformation is successful (if not, we need to retransform lol)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3682" data-index="33" style="margin-left:105px;"><span style="color:#344043"><span style="background-color:#FFFFFF">If plates are lawns.. we'll have to replate. Scrape some colonies and inoculate a 1mL LB-chlor culture. Replate them after 2-3 hours.</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3683" data-index="34" style="margin-left:105px;">If plates AREN'T lawns... Pick ~3 isolated colonies and do cPCR (use VF2 and VR. Size of plasmid should be 2.7kbp. Annealing T of VF2 and VR = 66C). After cPCR ends, run a gel to confirm the size of the PCR product and perform PCR cleanup. Send out for sequencing (use both forward and reverse, because the plasmid is so big). Grow 5 mL liquid cultures of the picked colonies and leave overnight (use 5 mL LB + 5 uL chlor media)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3684" data-index="35" style="margin-left:105px;">Don't forget to put them in the fridge after you're done!</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3685" data-index="36" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3686" data-index="37" style="margin-left:35px;">Redid transformation 9/20!! 4th time??</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3687" data-index="38" style="margin-left:35px;">9/21 - transformation successful. Sent sequencing, ran gels. Bands showed 1.2kb?? Still wrong size.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3688" data-index="39" style="margin-left:35px;">Trevor! suggested sequencing the DNA from the actual transformation kit. Will run gels/PCR cleanup tomorrow.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3689" data-index="40" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3690" data-index="41" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3691" data-index="42" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3692" data-index="43" style="margin-left:70px;"><b>DAY 3</b> Mini prep the overnight liquid cultures if the sequences are correct.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3693" data-index="44" style="margin-left:70px;">Get the linearized pSB1c3 backbone by doing PCR on the pabA+B minipreps. Use purple 1.5 mL "pSB1c3 backbone" primers in the box. Run PCR cleanup.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3694" data-index="45" style="margin-left:70px;">Gibson Assembly with pabC (with Gibson ends) and linearized pSB1c3 backbone and transform into 5-alpha.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3695" data-index="46" style="margin-left:105px;">NOTE: PCR tubes "p1 and p2" and "g1 and g2" are unknown ???? <span style="color:#344043"><span style="background-color:#FFFFFF">One set is the amplified pabC with ends for gibson.</span></span> So try gibsoning with both, but if you're not feelin it - try g1 and g2 tubes and we can troubleshoot from there. Both pairs of tubes should be in the box.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3696" data-index="47" style="margin-left:70px;"><b>DAY 4 </b>If Gibson fails and we can't successfully determine which PCR tubes contain the pabC with Gibson ends, then we have to remake it.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3697" data-index="48" style="margin-left:105px;">PCR the 1.5 mL tubes of pabC labeled "p2" and "p4". The primers for this PCR are labeled "pabC Gibson primer fwd/rev".</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3698" data-index="49" style="margin-left:70px;"><b>Alternative DAY 4 </b>If Gibson's successful!!!</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3699" data-index="50" style="margin-left:105px;">Do colony PCR and sequence (using VF2, VR or the pSB1c3 BB primers in the box). Run gel, do PCR cleanup.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3700" data-index="51" style="margin-left:105px;">Make liquid cultures of colonies with the right size (~2k bp)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3701" data-index="52" style="margin-left:70px;"><b>DAY 5 </b>Biobrick pabC to submit</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3702" data-index="53" style="margin-left:105px;">Biobricking process == Dilute miniprepped DNA to 25 ng/uL using MQ water. Put it in the foil tray..?? (Unclear what we put it in.) DNA needs to be dried, so leave it for 4-6 hours in laminar flow hood.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3703" data-index="54" style="margin-left:105px;">Actual instructions are google-able -- google "Biobrick submission" lol</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3704" data-index="55" style="margin-left:35px;">Make a construct with ABC using restriction digest. (Ask Trevor?? Ask if there are certain primers, enzymes etc. Look for protocol.) - <b><span style="color:#A6D461">I don't expect this to be done this week - so no worries</span></b></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3705" data-index="56" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3706" data-index="57" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3707" data-index="58" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3708" data-index="59" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3709" data-index="60" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3710" data-index="61" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3711" data-index="62" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-header2"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3712" data-index="63" style="margin-left:0;"><span style="color:#344043"><span style="background-color:#FFFFFF">Project 3: Overproduction of DAHP (enzyme in 1st committed step to making chorismate)</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3713" data-index="64" style="margin-left:35px;"><b><span style="color:#344043"><span style="background-color:#FFFFFF">DAY 1 </span></span></b><span style="color:#344043"><span style="background-color:#FFFFFF">Find where DAHP synthase liquid culture is lol</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3714" data-index="65" style="margin-left:70px;">Mike might know where it is</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3715" data-index="66" style="margin-left:70px;">??? Might have to regrow if can't be found</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3716" data-index="67" style="margin-left:105px;">Unclear what to do. Regrow from DAHP plates in 4C fridge. Otherwise if plates don't exist, bug eric lol</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3717" data-index="68" style="margin-left:35px;">DAHP synthase/chorismate ++ cell are found in a plastic gray box w/ yellow tape in the 4C fridge. 9/14/16</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3718" data-index="69" style="margin-left:35px;">Incubated DAHP liquid culture after picking from agar stab 9/14/16</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3719" data-index="70" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3720" data-index="71" style="margin-left:35px;">9/16 - made a DAHP plate for storage. Miniprep liquid culture and ran PCR using the DAHP primers to grab gene from psb1c3 plasmid. Sequenced.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3721" data-index="72" style="margin-left:35px;">9/20 - sequencing was good, will use with blitzen blue backbone</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3722" data-index="73" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3723" data-index="74" style="margin-left:35px;">Get the backbone from Blitzen Blue cells</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3724" data-index="75" style="margin-left:70px;"><b>DAY 1 </b>Pick 2 colonies from the blitzen blue plate. Make cultures - grow overnight in LB-chlor. <b><span style="color:#344043"><span style="color:#A6D461"><span style="background-color:#FFFFFF">I made LB-chlor plates with "blitzen blue 9/2 paba" on them by the RT shaker - use this plate.</span></span></span></b></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3725" data-index="76" style="margin-left:105px;">If it's a lawn - replate</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3726" data-index="77" style="margin-left:35px;">Incubation of blitzen blue culture (picked from plate) @11 am. 9/14/16</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3727" data-index="78" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3728" data-index="79" style="margin-left:70px;"><b>DAY 2</b> Miniprep Blitzen Blue cultures. Perform PCR using Blitzen Blue backbone primers which haven't been diluted yet. You'll need to dilute the ELIM DNA tubes labeled "bbbb" lol.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"><span><input checked="" disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3729" data-index="80" style="margin-left:105px;">I think there is a 1.5 mL white tube labeled "blitzen blue backbone 9/1/16" -- I forgot what it is. I think its a miniprep I did last week but was really low in concentration. Disregard this tube!</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3730" data-index="81" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span>●</span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3731" data-index="82" style="margin-left:35px;">9/20 realized blitzen blue is meant to be grown in LB-amp. Wondering why there are cells in my LB culture and what I miniprepped. We don't have time to figure that out, so I'm using Theresa/Cynthia's blitzen blue synthesized DNA plasmid</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3732" data-index="83" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3733" data-index="84" style="margin-left:35px;"><b>DAY after we find DAHP </b><span style="color:#344043"><span style="background-color:#FFFFFF">Put DAHP synthase gene into the chromoprotein vector</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3734" data-index="85" style="margin-left:70px;">Miniprep DAHP synthase culture and PCR with diluted primers in the box labeled "dahp".. think they're yellow/white 1.5mL tubes?</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3735" data-index="86" style="margin-left:70px;">Gibson two fragments together (DAHP + blitzen blue). Transform into 5alpha cells. PLATES SHOULD BE LB-AMP.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3736" data-index="87" style="margin-left:70px;">If successful, pick 2 colonies/grow culture</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3737" data-index="88" style="margin-left:70px;">Induce with IPTG, measure production.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3738" data-index="89" style="margin-left:35px;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3739" data-index="90" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-header2"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3740" data-index="91" style="margin-left:0;"><span style="color:#344043"><span style="background-color:#FFFFFF">Project 2: Knock out dihydropteroate synthase/<i>DHP</i> (gene that controls enzyme changing pABA to dihydropteroate) using CRISPRCas9</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3741" data-index="92" style="margin-left:35px;"><b>DAY 1 </b>Make LB +chlor/+folate plates</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3742" data-index="93" style="margin-left:70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Recipe for making LB chlor/folate plates can be googled. It should be </span></span><span style="color:#344043"><span style="background-color:#FFFFFF">~1.6mg/L.</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3743" data-index="94" style="margin-left:35px;">Basically follow <span style="color:#344043"><span style="background-color:#FFFFFF">https://www.addgene.org/static/data/07/52/50a35908-85d2-11e2-92ba-003048dd6500.pdf for your protocol. It's multiple steps so you don't have to feel pressured to complete them all??</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3744" data-index="95" style="margin-left:70px;"><b>DAY 1 </b>Digest pCas9 with BSA1 site, run gel, there should be a big band, do gel purification (run all 50 uL of it onto the gel! <span style="color:#344043"><span style="background-color:#FFFFFF">USE QIAGEN GEL EX KIT</span></span>). The undigest pcas9 plasmid t<span style="color:#000000">ube should be labeled "pcas9 478" in the box, use it for ur digestion!!</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3745" data-index="96" style="margin-left:70px;">Anneal oligos -<b><span style="color:#A6D461"> done already. Tube labeled "pcas9 anneal oligos".</span></b> Use 2 uL for the ligation step.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3746" data-index="97" style="margin-left:70px;"><b>DAY whatever - </b>Transform into NEB5alpha in (regular) LB-chlor plates</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3747" data-index="98" style="margin-left:35px;">If successful, perform replicates (LB-chlor and folate) - choose ones that die, because it means these cells are folate independent/the cells we want.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3748" data-index="99" style="margin-left:35px;">After cells have grown, m<span style="color:#344043"><span style="background-color:#FFFFFF">ake liquid cultures from these colonies and then run the assay the liquid cultures (accounting for opitcal density and stuff/aka they should be very similar)</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"><span><input disabled="" type="checkbox"/></span></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3749" data-index="100" style="margin-left:70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">We can perform thin layer chromatography using cell lysate/media.</span></span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3750" data-index="101" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3751" data-index="102" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3752" data-index="103" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3753" data-index="104" style="margin-left:0;">------------------</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-header1"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3754" data-index="105" style="margin-left:0;"><span style="color:#D10E0E">Additional Notes</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3755" data-index="106" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3756" data-index="107" style="margin-left:0;">ok so there are 3 (and 1/2) different subsubprojects on the aramid project right now</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3757" data-index="108" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3758" data-index="109" style="margin-left:0;">1- so we are trying to see if extra copies of pabA,B,C will increase the amount of paba we make</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3759" data-index="110" style="margin-left:0;">2-also if the knocking out the gene that controls enzyme changing pABA to dihydropteroate </div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3760" data-index="111" style="margin-left:0;">3- overproducing dahp (precursor to chorismate) </div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3761" data-index="112" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3762" data-index="113" style="margin-left:0;">1: (make sure to make 2 of each thing, check part 3 for deets)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3763" data-index="114" style="margin-left:0;">the biobrick pabA+B already has pabA and pabB in a construct - need to test amt</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3764" data-index="115" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3765" data-index="116" style="margin-left:0;">*I have already isolated the pabC from the E. coli and just have gibson left</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3766" data-index="117" style="margin-left:0;">-miniprep the 4 tubes from pabA+B, steal the backbone with PCR, then gibson</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3767" data-index="118" style="margin-left:35px;">-we will then get the pabC by itself in biobrick format</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3768" data-index="119" style="margin-left:35px;">-submit the biobrick</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3769" data-index="120" style="margin-left:35px;">-THEN: make a construct with ABC using the restriction digest</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3770" data-index="121" style="margin-left:0;">-----------------------</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3771" data-index="122" style="margin-left:0;">2:</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3772" data-index="123" style="margin-left:0;">crispr cas9 to knockout the gene</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3773" data-index="124" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3774" data-index="125" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3775" data-index="126" style="margin-left:0;">PROTOCOL: (if you need to do it again ask mike/trevor first, then jesica if all else fails)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3776" data-index="127" style="margin-left:0;">-----</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3777" data-index="128" style="margin-left:0;"><span style="color:#D10E0E">digest pCas9 with bsa1 site</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3778" data-index="129" style="margin-left:0;"><span style="color:#D10E0E">cut out the big vector of gel (10 kb)</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3779" data-index="130" style="margin-left:0;"><span style="color:#D10E0E">do gel purification on big band</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3780" data-index="131" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3781" data-index="132" style="margin-left:0;"><span style="color:#D10E0E">have my ordered oligos</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3782" data-index="133" style="margin-left:0;"><span style="color:#D10E0E">phosphorylate them (add phosphate to 5' end)</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3783" data-index="134" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3784" data-index="135" style="margin-left:0;"><span style="color:#D10E0E">then anneal</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3785" data-index="136" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3786" data-index="137" style="margin-left:0;"><span style="color:#D10E0E">-add 2 ul of annealed oligo</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3787" data-index="138" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3788" data-index="139" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3789" data-index="140" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3790" data-index="141" style="margin-left:0;"><span style="color:#D10E0E">transform into t7, neb5a, e. coli w</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3791" data-index="142" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3792" data-index="143" style="margin-left:0;"><span style="color:#D10E0E">p cas9 chlor resistant</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3793" data-index="144" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3794" data-index="145" style="margin-left:0;"><span style="color:#D10E0E">plate transformation</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3795" data-index="146" style="margin-left:0;"><span style="color:#D10E0E">do replicates</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3796" data-index="147" style="margin-left:0;"><span style="color:#D10E0E">choose ones that die!!</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3797" data-index="148" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3798" data-index="149" style="margin-left:0;"><span style="color:#D10E0E">---</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3799" data-index="150" style="margin-left:0;"><span style="color:#D10E0E">make +folate + chlor plates, regular chlor plates</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3800" data-index="151" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3801" data-index="152" style="margin-left:0;"><span style="color:#D10E0E">---</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3802" data-index="153" style="margin-left:0;"><span style="color:#D10E0E">after u get the compound (paba), thin layer chromatography to make sure. use cell lysate/cell culture</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3803" data-index="154" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3804" data-index="155" style="margin-left:0;"><span style="color:#D10E0E">--</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3805" data-index="156" style="margin-left:0;"><span style="color:#D10E0E">use culture samples with same optical density</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3806" data-index="157" style="margin-left:0;"><span style="color:#D10E0E">-----</span></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3807" data-index="158" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3808" data-index="159" style="margin-left:0;">what has been done is possibly all up until the transformation. <i> redo digestion/pcas9 + gex</i></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3809" data-index="160" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3810" data-index="161" style="margin-left:0;">need to plate transformation on folate + plates.</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3811" data-index="162" style="margin-left:0;">to make the folate + plates, try different concentrations, but first try with saturation. (ask charlie if need help?)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3812" data-index="163" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3813" data-index="164" style="margin-left:0;">after plating onto the folate + plates, then use the <b>velvet thing</b> to make a duplicate plate onto a REGULAR plate</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3814" data-index="165" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3815" data-index="166" style="margin-left:0;">-identify colonies that were unable to grow on the regular chlor, (-)folate plate, these are the successful transformations</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3816" data-index="167" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3817" data-index="168" style="margin-left:0;">- make liquid cultures from these colonies and then run the assay the liquid cultures (accounting for o-density and stuff)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3818" data-index="169" style="margin-left:35px;">check online for saturation ~1.6mg/L</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3819" data-index="170" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3820" data-index="171" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3821" data-index="172" style="margin-left:0;">3: dahp/chorismate overproduction (dahp synthase/chorismate ++)</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3822" data-index="173" style="margin-left:0;">-we already have the gene as part of a biobrick</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3823" data-index="174" style="margin-left:35px;">-<i>have pcr'ed out the gene </i>and we want to put it into the vector that came from the chromoproteins</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3824" data-index="175" style="margin-left:70px;">bb - backbone, bbbb = blitzen blue backbone primers</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3825" data-index="176" style="margin-left:70px;">regular BB = psb1c3 + temr + promotor</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3826" data-index="177" style="margin-left:35px;">-after, we will gibson the dahp synthase gene into the blitzen blue backbone</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3827" data-index="178" style="margin-left:35px;">-its IPTG inducible, so make sure to induce when we stick it in culture</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3828" data-index="179" style="margin-left:35px;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3829" data-index="180" style="margin-left:0;">-what the labwork actually is:</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3830" data-index="181" style="margin-left:35px;">-all primers are ordered</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3831" data-index="182" style="margin-left:70px;">-miniprep the blitzen blue backbone and then pcr with the bb bb primers</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:0;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3832" data-index="183" style="margin-left:0;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3833" data-index="184" style="margin-left:70px;">-miniprep the dahp synthase culture and then pcr with the primers</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3834" data-index="185" style="margin-left:70px;">-gibson the two fragments together</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3835" data-index="186" style="margin-left:70px;">-take the gibson stuff and transform it into cells and make sure to induce it!</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:105px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3836" data-index="187" style="margin-left:105px;">-measure production</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3837" data-index="188" style="margin-left:35px;">****-make two of every single liquid culture that was used for part 1</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3838" data-index="189" style="margin-left:70px;">-make one regular, other one make it electrocompetent, then transform in the gibson product and induce dahp synthase</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3839" data-index="190" style="margin-left:70px;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3840" data-index="191" style="margin-left:70px;">-measure and compare the differences with dahp overproduction</div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width:70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part3841" data-index="192" style="margin-left:70px;"></div></div></div><div class="mediocre-item is-text"><div class="mediocre-noteEditable multi-editable is-paragraph"><div 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Latest revision as of 19:48, 19 October 2016


Stanford-Brown 2016

pABA future · Benchling

pABA future

Made with Benchling
Project: iGEM 2016
Authors: Eric Liu, Anna Le
Date: 2016-08-30
Tuesday, 8/30
**First few steps of each project are definitely doable and prob wont take too much time!** Currently there are 3 subprojects under pABA. In the end we hope to consolidate these subprojects into one. Our subprojects are:
1.
Introducing extra copies of pab,A,B,C to increase pABA production
2.
Knocking out the last enzyme of the pathway to stop pABA from being further converted into folate
3.
Overproducing the first enzyme in the pathway
Inventory
ELIM DNA tubes marked with blue X's along bottom row = don't use
Pink 1.5 mL tubes with "25" on them = miniprepped pab(A+B) cultures
WHITE "Psb1c3 bb __ PCR 9/1" tubesand BLUE "9/2 1/2/1b/2b" tubes are trash lol.
"pcas9 478" tube = undigested pcas9 plasmid that needs to be digested for Project #2. Plasmid size is 10k
Primers
"psb1c3 backbone"
"c gibson" = primers for pabC to make gibson ends
"dahp" - primers for DAHP synthase gene to make gibson ends
pc9 PCR tube is probably useless.
General sizes
pcas9 = 10k
psb1c3 backbone (const. promoter + terminator) = 2.7kb
blitzen blue backbone = 4kb
dahp synthase = 1k
To do
Project 1: Extra copies of pabA,B,C to increase pABA production
Biobrick pabC
DAY 1 Transform PabA+B (DNA is in iGEM 2015 distribution kit #2) under 13P into NEB 5alpha cells. (Not making protein, so no need to use T7 cells. Also 5-alpha is more efficient anyways.) --- AL - I've done this already, I'm leaving the plates out over the weekend (9/3-9/5). They should be by the RT shaker)
Amy redid this 9/6 because 9/2 transformation failed.
Redid transformation 9/14.
Sequenced colonies, ran gels - gels did not have bands 2.7kb!! And sequencing results were trash as well!!
DAY 2 If transformation is successful (if not, we need to retransform lol)
If plates are lawns.. we'll have to replate. Scrape some colonies and inoculate a 1mL LB-chlor culture. Replate them after 2-3 hours.
If plates AREN'T lawns... Pick ~3 isolated colonies and do cPCR (use VF2 and VR. Size of plasmid should be 2.7kbp. Annealing T of VF2 and VR = 66C). After cPCR ends, run a gel to confirm the size of the PCR product and perform PCR cleanup. Send out for sequencing (use both forward and reverse, because the plasmid is so big). Grow 5 mL liquid cultures of the picked colonies and leave overnight (use 5 mL LB + 5 uL chlor media)
Don't forget to put them in the fridge after you're done!
Redid transformation 9/20!! 4th time??
9/21 - transformation successful. Sent sequencing, ran gels. Bands showed 1.2kb?? Still wrong size.
Trevor! suggested sequencing the DNA from the actual transformation kit. Will run gels/PCR cleanup tomorrow.
DAY 3 Mini prep the overnight liquid cultures if the sequences are correct.
Get the linearized pSB1c3 backbone by doing PCR on the pabA+B minipreps. Use purple 1.5 mL "pSB1c3 backbone" primers in the box. Run PCR cleanup.
Gibson Assembly with pabC (with Gibson ends) and linearized pSB1c3 backbone and transform into 5-alpha.
NOTE: PCR tubes "p1 and p2" and "g1 and g2" are unknown ???? One set is the amplified pabC with ends for gibson. So try gibsoning with both, but if you're not feelin it - try g1 and g2 tubes and we can troubleshoot from there. Both pairs of tubes should be in the box.
DAY 4 If Gibson fails and we can't successfully determine which PCR tubes contain the pabC with Gibson ends, then we have to remake it.
PCR the 1.5 mL tubes of pabC labeled "p2" and "p4". The primers for this PCR are labeled "pabC Gibson primer fwd/rev".
Alternative DAY 4 If Gibson's successful!!!
Do colony PCR and sequence (using VF2, VR or the pSB1c3 BB primers in the box). Run gel, do PCR cleanup.
Make liquid cultures of colonies with the right size (~2k bp)
DAY 5 Biobrick pabC to submit
Biobricking process == Dilute miniprepped DNA to 25 ng/uL using MQ water. Put it in the foil tray..?? (Unclear what we put it in.) DNA needs to be dried, so leave it for 4-6 hours in laminar flow hood.
Actual instructions are google-able -- google "Biobrick submission" lol
Make a construct with ABC using restriction digest. (Ask Trevor?? Ask if there are certain primers, enzymes etc. Look for protocol.) - I don't expect this to be done this week - so no worries
Project 3: Overproduction of DAHP (enzyme in 1st committed step to making chorismate)
DAY 1 Find where DAHP synthase liquid culture is lol
Mike might know where it is
??? Might have to regrow if can't be found
Unclear what to do. Regrow from DAHP plates in 4C fridge. Otherwise if plates don't exist, bug eric lol
DAHP synthase/chorismate ++ cell are found in a plastic gray box w/ yellow tape in the 4C fridge. 9/14/16
Incubated DAHP liquid culture after picking from agar stab 9/14/16
9/16 - made a DAHP plate for storage. Miniprep liquid culture and ran PCR using the DAHP primers to grab gene from psb1c3 plasmid. Sequenced.
9/20 - sequencing was good, will use with blitzen blue backbone
Get the backbone from Blitzen Blue cells
DAY 1 Pick 2 colonies from the blitzen blue plate. Make cultures - grow overnight in LB-chlor. I made LB-chlor plates with "blitzen blue 9/2 paba" on them by the RT shaker - use this plate.
If it's a lawn - replate
Incubation of blitzen blue culture (picked from plate) @11 am. 9/14/16
DAY 2 Miniprep Blitzen Blue cultures. Perform PCR using Blitzen Blue backbone primers which haven't been diluted yet. You'll need to dilute the ELIM DNA tubes labeled "bbbb" lol.
I think there is a 1.5 mL white tube labeled "blitzen blue backbone 9/1/16" -- I forgot what it is. I think its a miniprep I did last week but was really low in concentration. Disregard this tube!
9/20 realized blitzen blue is meant to be grown in LB-amp. Wondering why there are cells in my LB culture and what I miniprepped. We don't have time to figure that out, so I'm using Theresa/Cynthia's blitzen blue synthesized DNA plasmid
DAY after we find DAHP Put DAHP synthase gene into the chromoprotein vector
Miniprep DAHP synthase culture and PCR with diluted primers in the box labeled "dahp".. think they're yellow/white 1.5mL tubes?
Gibson two fragments together (DAHP + blitzen blue). Transform into 5alpha cells. PLATES SHOULD BE LB-AMP.
If successful, pick 2 colonies/grow culture
Induce with IPTG, measure production.
Project 2: Knock out dihydropteroate synthase/DHP (gene that controls enzyme changing pABA to dihydropteroate) using CRISPRCas9
DAY 1 Make LB +chlor/+folate plates
Recipe for making LB chlor/folate plates can be googled. It should be ~1.6mg/L.
Basically follow https://www.addgene.org/static/data/07/52/50a35908-85d2-11e2-92ba-003048dd6500.pdf for your protocol. It's multiple steps so you don't have to feel pressured to complete them all??
DAY 1 Digest pCas9 with BSA1 site, run gel, there should be a big band, do gel purification (run all 50 uL of it onto the gel! USE QIAGEN GEL EX KIT). The undigest pcas9 plasmid tube should be labeled "pcas9 478" in the box, use it for ur digestion!!
Anneal oligos - done already. Tube labeled "pcas9 anneal oligos". Use 2 uL for the ligation step.
DAY whatever - Transform into NEB5alpha in (regular) LB-chlor plates
If successful, perform replicates (LB-chlor and folate) - choose ones that die, because it means these cells are folate independent/the cells we want.
After cells have grown, make liquid cultures from these colonies and then run the assay the liquid cultures (accounting for opitcal density and stuff/aka they should be very similar)
We can perform thin layer chromatography using cell lysate/media.
------------------
Additional Notes
ok so there are 3 (and 1/2) different subsubprojects on the aramid project right now
1- so we are trying to see if extra copies of pabA,B,C will increase the amount of paba we make
2-also if the knocking out the gene that controls enzyme changing pABA to dihydropteroate
3- overproducing dahp (precursor to chorismate)
1: (make sure to make 2 of each thing, check part 3 for deets)
the biobrick pabA+B already has pabA and pabB in a construct - need to test amt
*I have already isolated the pabC from the E. coli and just have gibson left
-miniprep the 4 tubes from pabA+B, steal the backbone with PCR, then gibson
-we will then get the pabC by itself in biobrick format
-submit the biobrick
-THEN: make a construct with ABC using the restriction digest
-----------------------
2:
crispr cas9 to knockout the gene
PROTOCOL: (if you need to do it again ask mike/trevor first, then jesica if all else fails)
-----
digest pCas9 with bsa1 site
cut out the big vector of gel (10 kb)
do gel purification on big band
have my ordered oligos
phosphorylate them (add phosphate to 5' end)
then anneal
-add 2 ul of annealed oligo
transform into t7, neb5a, e. coli w
p cas9 chlor resistant
plate transformation
do replicates
choose ones that die!!
---
make +folate + chlor plates, regular chlor plates
---
after u get the compound (paba), thin layer chromatography to make sure. use cell lysate/cell culture
--
use culture samples with same optical density
-----
what has been done is possibly all up until the transformation. redo digestion/pcas9 + gex
need to plate transformation on folate + plates.
to make the folate + plates, try different concentrations, but first try with saturation. (ask charlie if need help?)
after plating onto the folate + plates, then use the velvet thing to make a duplicate plate onto a REGULAR plate
-identify colonies that were unable to grow on the regular chlor, (-)folate plate, these are the successful transformations
- make liquid cultures from these colonies and then run the assay the liquid cultures (accounting for o-density and stuff)
check online for saturation ~1.6mg/L
3: dahp/chorismate overproduction (dahp synthase/chorismate ++)
-we already have the gene as part of a biobrick
-have pcr'ed out the gene and we want to put it into the vector that came from the chromoproteins
bb - backbone, bbbb = blitzen blue backbone primers
regular BB = psb1c3 + temr + promotor
-after, we will gibson the dahp synthase gene into the blitzen blue backbone
-its IPTG inducible, so make sure to induce when we stick it in culture
-what the labwork actually is:
-all primers are ordered
-miniprep the blitzen blue backbone and then pcr with the bb bb primers
-miniprep the dahp synthase culture and then pcr with the primers
-gibson the two fragments together
-take the gibson stuff and transform it into cells and make sure to induce it!
-measure production
****-make two of every single liquid culture that was used for part 1
-make one regular, other one make it electrocompetent, then transform in the gibson product and induce dahp synthase
-measure and compare the differences with dahp overproduction