Difference between revisions of "Team:DTU-Denmark/Notebook"

Line 452: Line 452:
 
         <h2 class="h2">July</h2>
 
         <h2 class="h2">July</h2>
 
 
−
<!--WEEK2-->
+
<!--WEEK6-->
 
 
−
<!--WEEK3-->
+
<div class="panel panel-default">
 +
        <a data-toggle="collapse" href="#collapse6" aria-expanded="false" aria-controls="collapse6"> <!--change x2-->
 +
            <div class=" panel-heading" role="tab" id="heading6"> <!--change x1-->
 +
                <h4 class="panel-title">
 +
                    Week 6 (July 4 - July 10) <!-- TITLE -->
 +
                </h4>
 +
            </div>
 +
        </a>
 +
       
 +
        <!--CONTENT-->
 +
        <div id="collapse6" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading6"> <!-- change x2 -->
 +
            <div class="panel-body">
 +
                <h3>Wetlab</h3>
 +
               
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/a/ae/T--DTU-Denmark--moleculartools.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Molecular Toolbox</h5>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
                   
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/a/a7/T--DTU-Denmark--pex10.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>CRISPR-Cas9 induced <em>PEX10</em>knockout</h5>
 +
                                <p>Purification of pCRISPRyl plasmid from O/N cultures.  Successful digestion of CRISPRyl plasmid with restriction enzymes AatII and NdeI to verify the plasmid.
 +
                                Gibson Assembly of digested CRISPRyl plasmid and protospacers.
 +
</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/5/57/T--DTU-Denmark--ura3.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>CRISPR-Cas9 induced <em>URA3</em>insertion</h5>
 +
                                <p>Purification of pCRISPRyl. 
 +
                                Successful restriction analysis of pCRISPRyl with AatII and NdeI to verify the plasmid.
 +
                                Gibson assembly of pCRISPRyl and the hybridized sgRNAs targeting the <em>SUC2</em> gene. </p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/a/ac/T--DTU-Denmark--pSB1A8YL.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>pSB1A8YL</h5>
 +
                                <p>YES! finally a construct that seems to have the correct length! Both the analytical digestion and PCRs seems to confirm our construct.
 +
                                The construct was also sent for sequencing.
 +
                                We spent some time trying to figure out how to test the plasmid. We ended up retrieving the BBa_K592009, BBa_K592010, BBa_E1010 and BBa_J23110 parts from the distribution kit, and pair them. The idea is that if we are able to make a construct in our backbone, we should see a visual output.
 +
                                Unfortunately we were not able to retrieve the BBa_K592010 from the distribution kit, and it was decided to leave this for now.
 +
</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
        <!--/molecular toolbox-->       
 +
            <!-- /products -->
 +
                   
 +
                   
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/7/79/T--DTU-Denmark--substrate.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Substrates</h5>
 +
                            <p>Contamination of experiments was determined with microscopi. Might come from the minimal media being contaminated.
 +
                            First test on complex glycerol based media. <em>Y. lipolytica</em> seems to grow better than <em>Saccharomyces cerevisiae</em>.
 +
                            The growth form on different C-sources is analysed with microscopi. There seem to be different amounts of planktonic and filamentous growth depending on energy source.
 +
</p>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
            <!-- /substrate -->
 +
        <!-- /wetlab -->   
 +
               
 +
                <h3 style="clear:both;">Compute</h3>
 +
                 
 +
                    </div> <!-- /grid-row -->
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/1/1f/T--DTU-Denmark--software.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Software</h5>
 +
                            <p>Implementation of algorithm already started. The script is being written in Python3 with the intention to be easily modifiable so no external packages are needed although Anaconda is being used. </p>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
        <!-- /compute -->
 +
            </div> <!--/panel body-->
 +
        </div> <!--/collapse--> 
 +
    </div> <!--/panel-->
 
 
−
<!--WEEK4-->
+
<!--WEEK7-->
 
 
−
<!--WEEK5-->
+
<div class="panel panel-default">
 +
        <a data-toggle="collapse" href="#collapse7" aria-expanded="false" aria-controls="collapse7"> <!--change x2-->
 +
            <div class=" panel-heading" role="tab" id="heading1"> <!--change x7-->
 +
                <h4 class="panel-title">
 +
                    Week 7 (July 11 - July 17) <!-- TITLE -->
 +
                </h4>
 +
            </div>
 +
        </a>
 +
       
 +
        <!--CONTENT-->
 +
        <div id="collapse7" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading7"> <!-- change x2 -->
 +
            <div class="panel-body">
 +
                <h3>Wetlab</h3>
 +
               
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/a/ae/T--DTU-Denmark--moleculartools.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Molecular Toolbox</h5>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
                   
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/a/a7/T--DTU-Denmark--pex10.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>CRISPR-Cas9 induced <em>PEX10</em>knockout</h5>
 +
                                <p>The assembled CRISPRyl plasmid with <em>PEX10</em> protospacers was transformed in competent <em>E. coli</em> DH5α cells.
 +
                                Purification of assembled CRISPRyl plasmid with Pex10 protospacers.
 +
                                The assembled CRISPRyl plasmid with <em>PEX10</em> protospacers was digested with AvrII to test if the <em>PEX10</em> protospacer was inserted. The digestion was successful and showed no cutting by AvrII indicating correct insertion of <em>PEX10</em> protospacers.
 +
                                First attempt of transformation with CRISPRyl plasmid with <em>PEX10</em> protospacers in <em>Yarowia lipolytica</em> PO1f &Delta;<em>ku70</em>. The transformation was unsuccessful. There was growth all over the plates. </p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/5/57/T--DTU-Denmark--ura3.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>CRISPR-Cas9 induced <em>URA3</em>insertion</h5>
 +
                                <p>Confirmation of successful Gibson assembly through restriction analysis.
 +
                                First (unsuccessful) transformation of pCRISPRyl and pCRISPRyl+sgRNAs into <em>Yarowia lipolytica</em> PO1f &Delta;<em>ku70</em>.</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
        <!--/molecular toolbox-->       
 +
           
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/f/f1/T--DTU-Denmark--products.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Products</h5>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
                   
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/e/e2/T--DTU-Denmark--betacarotene.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>Beta-Carotene</h5>
 +
                                <p>BioBricks BBa_K152005 (<em>crtE</em>,<em>crtI</em>,<em>crtYB</em>,<em>gfp</em>) and BBa_K530002 (<em>crtI</em>) were received from the registry. E. coli with BBa_K152005 could only grow without Ampicilin, so we suspect a problem with the AmpR backbone. We decided not to use this biobrick and instead only work with BBa_K530002, BBa_K530000 and BBa_K530001.</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
                        <div class="grid-row">
 +
                            <div class="col-md-1 col-sm-1 col-xs-12"></div>
 +
                            <div class="col-md-2 col-sm-2 col-xs-12">
 +
                                <img class="lvlthree" src="https://static.igem.org/mediawiki/2016/a/ad/T--DTU-Denmark--proinsulin.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>Proinsulin</h5>
 +
                                <p>Designing and ordering synthetic gBlock from IDT encoding TEF1, proinsulin and GFP.</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 +
            <!-- /products -->
 +
                   
 +
                   
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/7/79/T--DTU-Denmark--substrate.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Substrates</h5>
 +
                            <p>Test shows no growth on samples of wastewater from areas contaminated with leftovers of organic molecules from fossil oil.
 +
                            Experiments with glycerol byproduct from Emmelev biodiesel production showed to be contaminated.
 +
                            We investigated where the contaminations could come from and a contaminated pipet seemed to be the source.
 +
</p>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
            <!-- /substrate -->
 +
        <!-- /wetlab -->   
 +
               
 +
                <h3 style="clear:both;">Compute</h3>
 +
                    <div class="grid-row">
 +
                        <div class="col-md-3 col-sm-3 col-xs-12">
 +
                            <img class="lvltwo" src="https://static.igem.org/mediawiki/2016/8/8d/T--DTU-Denmark--hardware.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Hardware</h5>
 +
                            <p>The library for our LCD Display is commented in Chinese. Shoutout to James for translating. Chris, our supervisor, brought us in contact with Martin and Erik, two professors from DTU Nano and Elektro. Our university is running a new project course in January where 1st semester students ferment their own cultures and build devices to monitor growth. They built a measuring chamber and a simple circuit, reading the voltage over the photodiode with a multimeter. Isn’t there a way to do all this using an Arduino? We set a meeting for next week.  Reading about digital to analog conversion(DAC) and the reverse(ADC). Dimming LEDs in the office</p>
 +
                        </div>
 +
                    </div> <!-- /grid-row -->
 +
        <!-- /compute -->
 +
            </div> <!--/panel body-->
 +
        </div> <!--/collapse--> 
 +
    </div> <!--/panel-->
 +
 +
<!--WEEK8-->
 +
 +
<!--WEEK9-->
 
 
 
         </div>
 
         </div>

Revision as of 13:19, 19 October 2016

New HTML template for the wiki




Bootstrap Example

Title

leader under the title, short introduction. Ubique moderatius efficiantur eum et, dico oporteat recusabo ius cu, pro id modus sadipscing. Maluisset patrioque eum ad, mel eius doctus accommodare eu, minimum deleniti repudiandae mel ea. Noster nostrud diceret sea no. Eos an nullam molestiae signiferumque, vel ne laudem ignota oblique. Duo te luptatum percipitur signiferumque, at dicunt iriure dolorem his.


June

Quote Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer posuere erat a ante.

Someone famous in Source Title

Wetlab

Yarowia lipolytica PO1f Δku70 was obtained from Cory M. Schwartz, cultivated and freeze stocked for future use.

Compute

Hardware

Our Arduino starter kits arrived! Make an LED blink. that’s how it begins.

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced PEX10knockout

Y. lipolytica PO1f genome sequence was annotated and protospacer for targeting PEX10 was designed and ordered from IDT. Protospacer for Gibson Assembly with CRISPRyl plasmid (Addgene plasmid #70007) SCR1'- tRNAGly (bold), Protospacer (underlined), sgRNA (italic) 5'-GGGTCGGCGCAGGTTGACGTGTACAAGGAGGAGCTGGAGAGTTTTAGAGCTAGAAATAGC-3' Oligos designed to amplify a 1kb region upstream and downstream PEX10 and anneal together by fusion PCR were also ordered from IDT.

CRISPR-Cas9 induced URA3insertion

The Y. lipolytica PO1f genome sequence was annotated and uploaded to Benchling for sgRNA design. sgRNAs targeting the SUC2 gene were designed. Primers were designed that amplify the functional URA3 gene including 1 kb upstream and downstream flanking regions.

pSB1A8YL

Ran a bunch of PCRs to amplify the pUC19 part of our plasmid, but it’s not working - nothing but smear. Tried to transform the pUC19 plasmid into Escherichia coli.

Substrates

We did an initial experiment determining the full growth cycle of Y. lipolytica W29. This will be used to plan and time the following growth experiments. Waste glycerol from the industrial biodiesel producer DAKA is acquired for late screening.

Compute

Hardware

We started building light sensors using photoresistors. Shortlisting ideas for our final project: - A microtiter plate reader - Hack a printer to build a membrane homogenizer - Chemostat bioreactor

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced PEX10knockout

Genomic DNA from Y. lipolytica PO1f Δku70 and Y. lipolytica W29 was purified. PEX10 flanking regions were successfully amplified from Y. lipolytica PO1f Δku70.

CRISPR-Cas9 induced URA3insertion

Genomic DNA from Y. lipolytica PO1f Δku70 and Y. lipolytica W29 was purified. - PCR attempts to amplify URA3 and flanks failed. - sgRNAs targeting the SUC2 gene were hybridized.

pSB1A8YL

Purified the plasmid from the transformants and use this as template for PCR, although it’s still not giving any bands.

Substrates

We did initial growth experiments on minimal media with an array of different carbon sources. This experiment was discarded due to lack of repeats and wrong vitamin solution for minimal media. Waste from canola oil production by Grønninggaard is acquired. Molasses from Dansukker sugar production is acquired.

Compute

Hardware

Exploring the Arduino IDE and all the electronic components we ordered. There is so much to learn.

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced URA3insertion

PCR attempts to amplify URA3 + flanks from Y. lipolytica W29 genomic DNA failed. New primers were ordered.

pSB1A8YL

We realized that the name of the primer had been mixed up! Now that the right primers are used, we get excellent bands on our gel… Guess you have to make the stupid mistakes in the beginning? The gBlock containing the other part of the plasmid also arrived. This also gives excellent bands on the gel when amplifying it by PCR. Ran the first USER and transformed E. coli cells. The transformants were left on the bench over the weekend

Substrates

We have data from the first successful growth experiment. Starch, Xylose, Arabinose, Maltose and Lactose are not suitable for Y. lipolytica fermentation. This will be repeated next week to make sure. Waste glycerol from the industrial biodiesel producer Perstop is acquired.

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced PEX10knockout

Received CRISPRyl plasmid (Addgene plasmid #70007). The procedure from Addgene was followed.

CRISPR-Cas9 induced URA3insertion

Successful amplification of URA3 + flanks from Y. lipolytica W29 genomic DNA. Because of low quality of the genomic DNA, the initial PCR product was taken for further amplification. Received CRISPRyl plasmid (Addgene plasmid #70007). The procedure from Addgene was followed.

pSB1A8YL

YES, colonies! colonies were picked and used for colony PCR, but it was not successful. We’ll just have to crank on! - Colonies from the same plates were re streaked and plasmids were purified from the resulting colonies. - Restriction analysis yielded weird bands. - This week passes restreaking colonies to yield pure colonies and trying to find the correct transformants through by purifying the plasmids and subjecting it to analytical digestion. So far no luck!

Substrates

Repeated positive results with growth on glucose, glycerol, fructose, sucrose and oil. Y. lipolytica should not be able to grow on sucrose. The experiments on starch, xylose, arabinose, Maltose and Lactose are still negative for Y. lipolytica. Waste glycerol from the industrial biodiesel producer Emmelev is acquired.

Compute

Genome Scale Modeling

Planning of Genome-scale modelling strategies began, decided to attempt media optimization using phenotype phase plane, team starts to research and learn FBA for GSM.

Software

Initiation of task by designing the workflow needed to achieve the final purpose of the software. Tasks agreed upon discussion : script in python , number and format proxy of the input files needed , restriction site implementation , development GUI, gui library for python (tkinter)

Hardware

Still playing.

July

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced PEX10knockout

Purification of pCRISPRyl plasmid from O/N cultures. Successful digestion of CRISPRyl plasmid with restriction enzymes AatII and NdeI to verify the plasmid. Gibson Assembly of digested CRISPRyl plasmid and protospacers.

CRISPR-Cas9 induced URA3insertion

Purification of pCRISPRyl. Successful restriction analysis of pCRISPRyl with AatII and NdeI to verify the plasmid. Gibson assembly of pCRISPRyl and the hybridized sgRNAs targeting the SUC2 gene.

pSB1A8YL

YES! finally a construct that seems to have the correct length! Both the analytical digestion and PCRs seems to confirm our construct. The construct was also sent for sequencing. We spent some time trying to figure out how to test the plasmid. We ended up retrieving the BBa_K592009, BBa_K592010, BBa_E1010 and BBa_J23110 parts from the distribution kit, and pair them. The idea is that if we are able to make a construct in our backbone, we should see a visual output. Unfortunately we were not able to retrieve the BBa_K592010 from the distribution kit, and it was decided to leave this for now.

Substrates

Contamination of experiments was determined with microscopi. Might come from the minimal media being contaminated. First test on complex glycerol based media. Y. lipolytica seems to grow better than Saccharomyces cerevisiae. The growth form on different C-sources is analysed with microscopi. There seem to be different amounts of planktonic and filamentous growth depending on energy source.

Compute

Software

Implementation of algorithm already started. The script is being written in Python3 with the intention to be easily modifiable so no external packages are needed although Anaconda is being used.

Wetlab

Molecular Toolbox
CRISPR-Cas9 induced PEX10knockout

The assembled CRISPRyl plasmid with PEX10 protospacers was transformed in competent E. coli DH5α cells. Purification of assembled CRISPRyl plasmid with Pex10 protospacers. The assembled CRISPRyl plasmid with PEX10 protospacers was digested with AvrII to test if the PEX10 protospacer was inserted. The digestion was successful and showed no cutting by AvrII indicating correct insertion of PEX10 protospacers. First attempt of transformation with CRISPRyl plasmid with PEX10 protospacers in Yarowia lipolytica PO1f Δku70. The transformation was unsuccessful. There was growth all over the plates.

CRISPR-Cas9 induced URA3insertion

Confirmation of successful Gibson assembly through restriction analysis. First (unsuccessful) transformation of pCRISPRyl and pCRISPRyl+sgRNAs into Yarowia lipolytica PO1f Δku70.

Products
Beta-Carotene

BioBricks BBa_K152005 (crtE,crtI,crtYB,gfp) and BBa_K530002 (crtI) were received from the registry. E. coli with BBa_K152005 could only grow without Ampicilin, so we suspect a problem with the AmpR backbone. We decided not to use this biobrick and instead only work with BBa_K530002, BBa_K530000 and BBa_K530001.

Proinsulin

Designing and ordering synthetic gBlock from IDT encoding TEF1, proinsulin and GFP.

Substrates

Test shows no growth on samples of wastewater from areas contaminated with leftovers of organic molecules from fossil oil. Experiments with glycerol byproduct from Emmelev biodiesel production showed to be contaminated. We investigated where the contaminations could come from and a contaminated pipet seemed to be the source.

Compute

Hardware

The library for our LCD Display is commented in Chinese. Shoutout to James for translating. Chris, our supervisor, brought us in contact with Martin and Erik, two professors from DTU Nano and Elektro. Our university is running a new project course in January where 1st semester students ferment their own cultures and build devices to monitor growth. They built a measuring chamber and a simple circuit, reading the voltage over the photodiode with a multimeter. Isn’t there a way to do all this using an Arduino? We set a meeting for next week. Reading about digital to analog conversion(DAC) and the reverse(ADC). Dimming LEDs in the office

August

September

October

  • FIND US AT:
Facebook Twitter
  • DTU BIOBUILDERS
  • DENMARK
  • DTU - SØLTOFTS PLADS, BYGN. 221/002
  • 2800 KGS. LYNGBY

  • E-mail:
  • dtu-biobuilders-2016@googlegroups.com
  • MAIN SPONSORS:
Lundbeck fundation DTU blue dot Lundbeck fundation Lundbeck fundation