Team:WashU/Week12

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<h1>Crocin Extraction</h1>
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To accurately verify that our construct is indeed producing zeaxanthin, spectrophotometry must be used to characterize our biobrick part. To this end, we tested crocin in two different solvents, along with our Z^2 construct, compared to the absorption spectrum of zeaxanthin in hexane.
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http://dl.dropbox.com/u/88390549/CrocinGraphs.png
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This indicates that our Z^2 construct isn't actually producing crocin, or if it is, the crocin isn't expressed highly enough to absorb significantly.
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<h1>Saffron in a Kan</h1>
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We also PCR'd the Z construct in the submission plasmid, Psb1c3. We then digested the results with both EcoRI and PstI, as shown below, and saw that the PCR had indeed succeded.<br>
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http://lh6.googleusercontent.com/-7oeDkDT2Ef8/UClEy74B65I/AAAAAAAAAgI/_8myIyOxHdI/s800/zeeparts.jpg
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Finally, we ran an SDS-PAGE gel of our Z construct with a histidine tag in order to see the proteins produced using an extraction protocol from Sigma found here [INSERT PROTOCOL].
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<img src="https://static.igem.org/mediawiki/igem.org/f/f8/Zcdtopo_confirm.jpg">
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Performed another extraction using His-columns to purify ZCD with His tag and UGTCS2 with His tag and ran them on a SDS-Page gel.
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PCR of TOPO with Z and U again using colonies as template.
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In vitro assay for crocin production repeated with purified protein from extraction mentioned above.
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Digest of PsbA2 PCR'd with ZCD and control TOPO vector - cut with E and P
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Ligation of the above products with the chloramphenicol plasmid.
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We started with a transformation of the ligation yesterday using GC10s and 5 microliters of ligation mixture. Then, we plated the transformed cells in 100 microliter aliquots.
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Next, we ran a PCR of Z + Thiofusion using KlenTaq at three different temperatures (45, 50, and 55 degrees) in order to figure out why the PCR isn't working. <html>
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<img src="https://static.igem.org/mediawiki/2012/0/05/0.png">
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We also attempted to perform a zeaxanthin extraction using our culture of the <i>Synechocystis</i> PAL mutant.
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<table style="width:auto;"><tr><td><a href="http://picasaweb.google.com/lh/photo/2iExq1RPSgh9PGJVACqNsT5bkQKfZwcjUCwEKVIJGfc?feat=embedwebsite"><img src="http://lh4.googleusercontent.com/-5UrHwTMRPiM/UCvyrIlsayI/AAAAAAAAAhc/CKYZHdMvoFo/s640/P1060127.JPG" height="428" width="640" /></a></td></tr><tr><td style="font-family:arial,sans-serif; font-size:11px; text-align:right">From <a href="http://picasaweb.google.com/100308169786596215568/August152012?authuser=0&authkey=Gv1sRgCK2xxviFoP6ySA&feat=embedwebsite">August 15, 2012</a></td></tr></table>
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Since <i>Synechocystis</i> is a photoautotroph, it produces chlorophyll, resulting in the green color. The cyanobacterium also produces beta carotene, the precursor to zeaxanthin, which is the precursor to the crocin we are trying to produce. The peaks for beta carotene and zeaxanthin overlap, so resolving the difference may be difficult.
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SDS-PAGE gel of ZCD and UGTCS2 <i>E. coli</i> cultures (both induced at 20 degrees and non-induced) in order to see how much protein is produced from our cultures. TO do this, had to sonicate cultures. Potential issue: used LB to sonicate instead of proper sonication buffer.
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We also started a large culture of Z^2 (our culture of double transformed <i> E. coli </i> with both ZCD and the zeaxanthin-producing construct. We will use this culture to test the optimum conditions of inducer (arabinose and IPTG).
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KlenTaq PCR of Z construct with TOPO in order to eventually submit this part, regular Taq PCR of U construct with TOPO because we are running low on KlenTaq
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Trypsin digestion of ZCD, UGTCS2, Z-construct, and a control<br>
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<img src="http://lh6.googleusercontent.com/-9EHSF2-bvAo/UC54AJNrE4I/AAAAAAAAAhw/kDoPb-XA_h0/s640/induced%2520SDS.png"><br>
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We performed a digest of Z-TOPO and U-TOPO from yesterday, followed by ligation of Z + TOPO, U + TOPO, and our main construct CS42S for submission.
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We then transformed our cells with ligation results and plated them.
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Finally, we cleaned up our lab and moved items into storage. :(
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On our last day, we spent time working on the dry lab stuff and getting our DNA ready for submission and sequencing.
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[https://2012.igem.org/Team:WashU/WeeklyLog Back to Weekly Log]
[https://2012.igem.org/Team:WashU/WeeklyLog Back to Weekly Log]

Latest revision as of 02:26, 15 September 2012




Monday, August 13

Crocin Extraction

To accurately verify that our construct is indeed producing zeaxanthin, spectrophotometry must be used to characterize our biobrick part. To this end, we tested crocin in two different solvents, along with our Z^2 construct, compared to the absorption spectrum of zeaxanthin in hexane.

http://dl.dropbox.com/u/88390549/CrocinGraphs.png

This indicates that our Z^2 construct isn't actually producing crocin, or if it is, the crocin isn't expressed highly enough to absorb significantly.

Saffron in a Kan

We also PCR'd the Z construct in the submission plasmid, Psb1c3. We then digested the results with both EcoRI and PstI, as shown below, and saw that the PCR had indeed succeded.
http://lh6.googleusercontent.com/-7oeDkDT2Ef8/UClEy74B65I/AAAAAAAAAgI/_8myIyOxHdI/s800/zeeparts.jpg

Finally, we ran an SDS-PAGE gel of our Z construct with a histidine tag in order to see the proteins produced using an extraction protocol from Sigma found here [INSERT PROTOCOL].
Tuesday, August 14

Performed another extraction using His-columns to purify ZCD with His tag and UGTCS2 with His tag and ran them on a SDS-Page gel.

PCR of TOPO with Z and U again using colonies as template.

In vitro assay for crocin production repeated with purified protein from extraction mentioned above.

Digest of PsbA2 PCR'd with ZCD and control TOPO vector - cut with E and P Ligation of the above products with the chloramphenicol plasmid.



Wednesday, August 15

We started with a transformation of the ligation yesterday using GC10s and 5 microliters of ligation mixture. Then, we plated the transformed cells in 100 microliter aliquots.

Next, we ran a PCR of Z + Thiofusion using KlenTaq at three different temperatures (45, 50, and 55 degrees) in order to figure out why the PCR isn't working.

We also attempted to perform a zeaxanthin extraction using our culture of the Synechocystis PAL mutant.

From August 15, 2012
Since Synechocystis is a photoautotroph, it produces chlorophyll, resulting in the green color. The cyanobacterium also produces beta carotene, the precursor to zeaxanthin, which is the precursor to the crocin we are trying to produce. The peaks for beta carotene and zeaxanthin overlap, so resolving the difference may be difficult.

SDS-PAGE gel of ZCD and UGTCS2 E. coli cultures (both induced at 20 degrees and non-induced) in order to see how much protein is produced from our cultures. TO do this, had to sonicate cultures. Potential issue: used LB to sonicate instead of proper sonication buffer.

We also started a large culture of Z^2 (our culture of double transformed E. coli with both ZCD and the zeaxanthin-producing construct. We will use this culture to test the optimum conditions of inducer (arabinose and IPTG).

KlenTaq PCR of Z construct with TOPO in order to eventually submit this part, regular Taq PCR of U construct with TOPO because we are running low on KlenTaq



Thursday, August 16

Trypsin digestion of ZCD, UGTCS2, Z-construct, and a control

We performed a digest of Z-TOPO and U-TOPO from yesterday, followed by ligation of Z + TOPO, U + TOPO, and our main construct CS42S for submission. We then transformed our cells with ligation results and plated them.

Finally, we cleaned up our lab and moved items into storage. :(


Friday, August 17

On our last day, we spent time working on the dry lab stuff and getting our DNA ready for submission and sequencing.

Retrieved from "http://2012.igem.org/Team:WashU/Week12"