Difference between revisions of "Team:Pittsburgh/Notebook"
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<h1 style="clear:both;">Week 2: May 31 - June 3</h1> | <h1 style="clear:both;">Week 2: May 31 - June 3</h1> | ||
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;"> | <img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;"> | ||
| − | < | + | <div class="summary" style="padding:5px;"> |
| + | <ul> | ||
<li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">efficiency</a> of competent cells</li> | <li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">efficiency</a> of competent cells</li> | ||
</ul> | </ul> | ||
| − | + | ||
<h3>Cell-Free Extract</h3> | <h3>Cell-Free Extract</h3> | ||
<ul><li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract reaction</a> with T7-GFP plasmid</li></ul> | <ul><li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract reaction</a> with T7-GFP plasmid</li></ul> | ||
| − | + | </div> | |
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;"> | <img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;"> | ||
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<li>Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA</li> | <li>Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA</li> | ||
</ul> | </ul> | ||
| − | + | ||
| − | + | ||
<h3>Toehold Switch</h3> | <h3>Toehold Switch</h3> | ||
<ul> | <ul> | ||
<li>Collins triggers activate the switches (both in plasmid form) to express LacZ</li> | <li>Collins triggers activate the switches (both in plasmid form) to express LacZ</li> | ||
</ul> | </ul> | ||
| − | + | ||
| − | + | ||
<h3>Reporter</h3> | <h3>Reporter</h3> | ||
<ul> | <ul> | ||
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<ul> | <ul> | ||
<li>Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA</li> | <li>Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>Toehold Switch</h3> | <h3>Toehold Switch</h3> | ||
<ul> | <ul> | ||
<li>Collins plasmids express LacZ with 25 ng of switch</li> | <li>Collins plasmids express LacZ with 25 ng of switch</li> | ||
<li>DNA oligos trigger Collins switches</li> | <li>DNA oligos trigger Collins switches</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>Reporter</h3> | <h3>Reporter</h3> | ||
<ul> | <ul> | ||
| − | <li>Identify successful ligations to terminator for amilCP and eGFP | + | <li>Identify successful ligations to terminator for amilCP and eGFP constructs using a <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#agarosegel" target="_blank">gel</a></li> |
<li>Send correct plasmids for <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#sequencing" target="_blank">sequencing</a> for confirmation</li> | <li>Send correct plasmids for <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#sequencing" target="_blank">sequencing</a> for confirmation</li> | ||
<li>Test plasmids in <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract</a></li> | <li>Test plasmids in <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract</a></li> | ||
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<ul> | <ul> | ||
<li>384-well plate requires at least 10 μL of reaction</li> | <li>384-well plate requires at least 10 μL of reaction</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>DNAzyme</h3> | <h3>DNAzyme</h3> | ||
<ul> | <ul> | ||
<li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#annealing" target="_blank">Anneal</a> PO strand with catalytic strand, both with and without erbium</li> | <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#annealing" target="_blank">Anneal</a> PO strand with catalytic strand, both with and without erbium</li> | ||
<li>Test success of annealing reaction in cell-free extract and with acrylamide gels</li> | <li>Test success of annealing reaction in cell-free extract and with acrylamide gels</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>Reporter</h3> | <h3>Reporter</h3> | ||
<ul> | <ul> | ||
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<ul> | <ul> | ||
<li>Linear eGFP construct does not produce a stronger signal than its plasmid form</li> | <li>Linear eGFP construct does not produce a stronger signal than its plasmid form</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>DNAzyme</h3> | <h3>DNAzyme</h3> | ||
<ul> | <ul> | ||
<li>DNAzyme duplex does not trigger toehold switch</li> | <li>DNAzyme duplex does not trigger toehold switch</li> | ||
<li>Erbium cleaves the P substrate strand</li> | <li>Erbium cleaves the P substrate strand</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>Reporter</h3> | <h3>Reporter</h3> | ||
<ul> | <ul> | ||
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<ul> | <ul> | ||
<li>Reactions can be diluted by one-half and still produce visible results in two hours</li> | <li>Reactions can be diluted by one-half and still produce visible results in two hours</li> | ||
| − | </ul | + | </ul> |
| − | + | ||
<h3>DNAzyme</h3> | <h3>DNAzyme</h3> | ||
<ul> | <ul> | ||
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<li>Six-hour time course of cleavage does not yield much additional information</li> | <li>Six-hour time course of cleavage does not yield much additional information</li> | ||
<li>Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates</li> | <li>Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates</li> | ||
| − | </ul | + | </ul> |
| − | + | <h3>Reporter</h3> | |
<ul> | <ul> | ||
<li>Continue amilCP cloning process</li> | <li>Continue amilCP cloning process</li> | ||
Revision as of 15:28, 1 August 2016
Contact Us
Thank you to our sponsors!
Our weekly progress. For a list of our protocols, visit the Protocols page
Contents
Week 1: May 23 - May 27
- Training begins
- Grow Top 10 competent cells.
- Brainstorm genetic circuits for a thallium sensor
- Lab safety training
Week 2: May 31 - June 3
- Test efficiency of competent cells
Cell-Free Extract
- Test cell-free extract reaction with T7-GFP plasmid
- Contact museums and summer programs for outreach opportunities
- Lab safety training
Week 3: June 6 - June 12
Reporter
- Contact museums and summer programs for outreach opportunities
Week 4: June 13 - June 17
Reporter
- Transform T7-GFP plasmid, lacZ alpha fragment, and eGFP
- Send promising T7 promoter -- amilCP ligations to be sequenced
- Perform double digest of T7 promoter and terminator from last week
- Ligate double-digested T7 promoter to new reporters (lacZ and eGFP)
- TECBio, DiSCoBio, and Tissue Engineering Camp outreach opportunities set
Week 5: June 20 - June 26
Cell-Free Extract
- Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA
Toehold Switch
- Collins triggers activate the switches (both in plasmid form) to express LacZ
Reporter
- Reach out to teams to collaborate based on last year's projects
Week 6: June 27 - July 3
Cell-Free Extract
- Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA
Toehold Switch
- Collins plasmids express LacZ with 25 ng of switch
- DNA oligos trigger Collins switches
Reporter
- Identify successful ligations to terminator for amilCP and eGFP constructs using a gel
- Send correct plasmids for sequencing for confirmation
- Test plasmids in cell-free extract
- amilCP does not produce color in cell-free reaction
- eGFP produces fluorescence comparable to that from the Collins T7-GFP plasmid in cell-free reaction
- Linearized plasmids containing only the promoter and insert (no terminator) do not express protein
- Work on outreach presentation for tissue engineering camp
Week 7: July 5 - July 8
Cell-Free Extract
- 384-well plate requires at least 10 μL of reaction
DNAzyme
- Anneal PO strand with catalytic strand, both with and without erbium
- Test success of annealing reaction in cell-free extract and with acrylamide gels
Reporter
- Sequenced amilCP construct does not contain amilCP
- Unsuccessfully linearize and amplify eGFP construct using PCR
- Practice outreach presentation for tissue engineering camp
- Develop DNAzymes for other heavy metals
Week 8: July 11 - July 17
Cell-Free Extract
- Linear eGFP construct does not produce a stronger signal than its plasmid form
DNAzyme
- DNAzyme duplex does not trigger toehold switch
- Erbium cleaves the P substrate strand
Reporter
- Restart amilCP cloning process
- First presentation at Camp BioE
- Prepare for UMD Mid-Atlantic Meet-Up
- Contact PLSG and NEB for sponsorship
Week 9: July 18 - July 22
Cell-Free Extract
- Reactions can be diluted by one-half and still produce visible results in two hours
DNAzyme
- dPAGE assay of P substrate cleavage suggests that the DNAzyme works, but results are not definitive
- Six-hour time course of cleavage does not yield much additional information
- Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates
Reporter
- Continue amilCP cloning process
- Clone RBS-T3 RNA polymerase to add into other constructs
- Presentation at Camp BioE
- UMD Mid-Atlantic Meet-Up
- Continue fundraising
- Discuss systems to model
