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

Line 453: Line 453:
 
 
 
<!--WEEK6-->
 
<!--WEEK6-->
 +
 +
 +
 +
<!--WEEK7-->
 +
 +
 +
 +
<!--WEEK8-->
 
 
 
<div class="panel panel-default">
 
<div class="panel panel-default">
−
        <a data-toggle="collapse" href="#collapse6" aria-expanded="false" aria-controls="collapse6"> <!--change x2-->
+
        <a data-toggle="collapse" href="#collapse8" aria-expanded="false" aria-controls="collapse8"> <!--change x2-->
−
            <div class=" panel-heading" role="tab" id="heading6"> <!--change x1-->
+
            <div class=" panel-heading" role="tab" id="heading8"> <!--change x1-->
 
                <h4 class="panel-title">
 
                <h4 class="panel-title">
−
                    Week 6 (July 4 - July 10) <!-- TITLE -->
+
                    Week 8 (July 18 - July 24) <!-- TITLE -->
 
                </h4>
 
                </h4>
 
            </div>
 
            </div>
Line 464: Line 472:
 
          
 
          
 
        <!--CONTENT-->
 
        <!--CONTENT-->
−
        <div id="collapse6" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading6"> <!-- change x2 -->
+
        <div id="collapse8" class="panel-collapse collapse" role="tabpanel" aria-labelledby="heading8"> <!-- change x2 -->
 
            <div class="panel-body">
 
            <div class="panel-body">
 
                <h3>Wetlab</h3>
 
                <h3>Wetlab</h3>
Line 484: Line 492:
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                                <h5>CRISPR-Cas9 induced <em>PEX10</em>knockout</h5>
 
                                <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.  
+
                                <p>Transformation in <em>Y. lipolytica</em> was repeated using the same transformation protocol from last week. However, this time less cells were used for the transformation. This did not work.</p>
−
                                Gibson Assembly of digested CRISPRyl plasmid and protospacers.  
+
−
</p>
+
 
                            </div>
 
                            </div>
 
                        </div> <!-- /grid-row -->
 
                        </div> <!-- /grid-row -->
Line 496: Line 502:
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                                <h5>CRISPR-Cas9 induced <em>URA3</em>insertion</h5>
 
                                <h5>CRISPR-Cas9 induced <em>URA3</em>insertion</h5>
−
                                <p>Purification of pCRISPRyl.
+
                                <p>Gibson assembly of sgRNAs targeting <em>SUC2</em> into pCRISPRyl.
−
                                Successful restriction analysis of pCRISPRyl with AatII and NdeI to verify the plasmid.
+
                                More (unsuccessful) transformations of pCRISPRyl and pCRISPRyl+sgRNAs into <em>Yarowia lipolytica</em> PO1f &Delta;<em>ku70</em>.</p>
−
                                Gibson assembly of pCRISPRyl and the hybridized sgRNAs targeting the <em>SUC2</em> gene. </p>
+
 
                            </div>
 
                            </div>
 
                        </div> <!-- /grid-row -->
 
                        </div> <!-- /grid-row -->
Line 508: Line 513:
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                            <div class="col-md-9 col-sm-9 col-xs-12">
 
                                <h5>pSB1A8YL</h5>
 
                                <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.
+
                                <p>The promoter was paired with the chromoproteins using 3A assembly. Unfortunately, no color was observed even though PCR and analytical digestion showed that the length of the construct are correct.</p>
−
                                The construct was also sent for sequencing.
+
                            </div>
−
                                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.
+
                        </div> <!-- /grid-row -->
−
                                Unfortunately we were not able to retrieve the BBa_K592010 from the distribution kit, and it was decided to leave this for now.
+
                        <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/f/f9/T--DTU-Denmark--promoter.png" alt="">
 +
                            </div>
 +
                            <div class="col-md-9 col-sm-9 col-xs-12">
 +
                                <h5>Promoters</h5>
 +
                                <p>TEF1 was amplified from gBlock by PCR.
 +
                                SCR1’-tRNA promoter was amplified from the pCRISPRyl plasmid by PCR with primers introducing a base substitution to remove illegal restriction site.
 
</p>
 
</p>
 
                            </div>
 
                            </div>
 
                        </div> <!-- /grid-row -->
 
                        </div> <!-- /grid-row -->
 
        <!--/molecular toolbox-->         
 
        <!--/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_K530000 (<em>crtYB</em>) and BBa_K530001 (<em>crtE</em>) from the distribution kit was successfully obtained.
 +
                                We designed Gibson primers including 5'-CACA-3' upstream of each start codon for assembly of all three genes.
 +
</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>3A assembly of the proinsulin gBlock and pSB1C3. Electrophoresis did not confirm presence of desire plasmid.</p>
 +
                            </div>
 +
                        </div> <!-- /grid-row -->
 
            <!-- /products -->
 
            <!-- /products -->
 
                      
 
                      
Line 525: Line 570:
 
                        <div class="col-md-9 col-sm-9 col-xs-12">
 
                        <div class="col-md-9 col-sm-9 col-xs-12">
 
                            <h5>Substrates</h5>
 
                            <h5>Substrates</h5>
−
                            <p>Contamination of experiments was determined with microscopi. Might come from the minimal media being contaminated.
+
                            <p><em>S. cerevisiae</em> does not grow as well as <em>Y. lipolytica</em> on glycerol based waste, but has an advantage on sucrose based ones. </p>
−
                            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>
 
                    </div> <!-- /grid-row -->
 
                    </div> <!-- /grid-row -->
Line 535: Line 577:
 
                  
 
                  
 
                <h3 style="clear:both;">Compute</h3>
 
                <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/d/d8/T--DTU-Denmark--modeling.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Genome Scale Modeling</h5>
 +
                            <p>Ben has kickstarted the modeling, by introducing the phenotype phase plane concept to the team, which is an extension to flux-balance analysis. The hope is that it will be possible to find optimal in-flow of various substrates in order to maximize product, in this case beta-carotene. Downloaded first <em>Y. lipolytica</em> model “MODEL1510060001”. Tutorials are studied in Matlab.
 +
</p>
 +
                        </div>
 
                    </div> <!-- /grid-row -->
 
                    </div> <!-- /grid-row -->
 
                    <div class="grid-row">
 
                    <div class="grid-row">
Line 543: Line 594:
 
                        <div class="col-md-9 col-sm-9 col-xs-12">
 
                        <div class="col-md-9 col-sm-9 col-xs-12">
 
                            <h5>Software</h5>
 
                            <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>
+
                            <p>Script writing is finished. Testing of functionality was initiated in order to find the most “sustainable” solution in terms of resources and the optimization step (reverse-translation) from the desired sequences to protein sequences. Approach decided for main body of script is building and checking the sequences “on the fly” while using as initial files the y_lip.txt (gcn), desired.fsa(protein sequences), ressites.txt(restriction sites).</p>
 +
                        </div>
 +
                    </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/2016/8/8d/T--DTU-Denmark--hardware.png" alt="">
 +
                        </div>
 +
                        <div class="col-md-9 col-sm-9 col-xs-12">
 +
                            <h5>Hardware</h5>
 +
                            <p>Successful tests at DTU Nano. We can measure growth of S. cerevisiae. Erik found a great photodetector that includes an amplifying circuit.</p>
 
                        </div>
 
                        </div>
 
                    </div> <!-- /grid-row -->
 
                    </div> <!-- /grid-row -->
Line 550: Line 610:
 
        </div> <!--/collapse-->   
 
        </div> <!--/collapse-->   
 
    </div> <!--/panel-->
 
    </div> <!--/panel-->
−
 
−
<!--WEEK7-->
 
−
 
−
 
−
 
−
<!--WEEK8-->
 
 
 
 
<!--WEEK9-->
 
<!--WEEK9-->

Revision as of 13:33, 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

Transformation in Y. lipolytica was repeated using the same transformation protocol from last week. However, this time less cells were used for the transformation. This did not work.

CRISPR-Cas9 induced URA3insertion

Gibson assembly of sgRNAs targeting SUC2 into pCRISPRyl. More (unsuccessful) transformations of pCRISPRyl and pCRISPRyl+sgRNAs into Yarowia lipolytica PO1f Δku70.

pSB1A8YL

The promoter was paired with the chromoproteins using 3A assembly. Unfortunately, no color was observed even though PCR and analytical digestion showed that the length of the construct are correct.

Promoters

TEF1 was amplified from gBlock by PCR. SCR1’-tRNA promoter was amplified from the pCRISPRyl plasmid by PCR with primers introducing a base substitution to remove illegal restriction site.

Products
Beta-Carotene

Biobricks BBa_K530000 (crtYB) and BBa_K530001 (crtE) from the distribution kit was successfully obtained. We designed Gibson primers including 5'-CACA-3' upstream of each start codon for assembly of all three genes.

Proinsulin

3A assembly of the proinsulin gBlock and pSB1C3. Electrophoresis did not confirm presence of desire plasmid.

Substrates

S. cerevisiae does not grow as well as Y. lipolytica on glycerol based waste, but has an advantage on sucrose based ones.

Compute

Genome Scale Modeling

Ben has kickstarted the modeling, by introducing the phenotype phase plane concept to the team, which is an extension to flux-balance analysis. The hope is that it will be possible to find optimal in-flow of various substrates in order to maximize product, in this case beta-carotene. Downloaded first Y. lipolytica model “MODEL1510060001”. Tutorials are studied in Matlab.

Software

Script writing is finished. Testing of functionality was initiated in order to find the most “sustainable” solution in terms of resources and the optimization step (reverse-translation) from the desired sequences to protein sequences. Approach decided for main body of script is building and checking the sequences “on the fly” while using as initial files the y_lip.txt (gcn), desired.fsa(protein sequences), ressites.txt(restriction sites).

Hardware

Successful tests at DTU Nano. We can measure growth of S. cerevisiae. Erik found a great photodetector that includes an amplifying circuit.

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