Team:WashU/Week8

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Revision as of 19:23, 19 July 2012

Monday, July 16

We ran multiple digests today, following the standard biobrick assembly protocol.

Digestions
Z and C: digest with P and E
Ligation 2: digest with X and P
Ligation 2: digest with X
Z and C: digest with E
ZCD: digest with X and P
UGTCs2: digest with X and P
2: digest with P
2: digest with B

The gel of these digests is found below.

In addition, we ran several PCRs today. Following the NEB protocol, we ran four PCRs to amplify the CCD plasmid (PUT PROPER NAME HERE) and also tried four colony PCRs, using colonies 2, 4, 13 and ______.

To enable amplification of our plasmid PSL2131, PsbA2, and CS42S, we designed primers to multiply the aforementioned DNA pieces.

We have started cultures of E. coli doubly transformed with our Z construct and CS42S at different chloramphenicol concentrations. We will miniprep them after they grow to obtain the DNA.
Tuesday, July 17

We repeated the digests and PCRs from yesterday in order to ascertain what went wrong. The PCRs yielded primer dimers and were thus unsuccessful, so we plan on troubleshooting our procedure to determine why our reactions are failing. [PICTURE OF GEL]

In order to increase our output of carotenoids in Synechocystis, we began a PAL mutant liquid culture of the cyanobacterium in order to transform with in the next few days.

Wednesday, July 18
This morning, we miniprepped a culture of PC42, our ligation of plasmid PSL2131 and construct CS42S. Then, we ran a gel of double and single digests of PC42, cut with X and P and just P, along with an uncut PC42 as a control. [GEL SHOWN BELOW]

In addition, we took the cultures of the double digest from Monday, miniprepped them, nanodropped, and then digested them with EcoRI and PstI. The digestions were then run on a gel [PICTURE BELOW]

Finally, we conducted an experiment to see if the copy number of a plasmid could be regulated by varying the antibiotic concentration. To do this we grew our E. coli which was transformed with both the zeaxanthin producing construct and our Cs42s construct with varying amounts of chloramphocol while holding ampicilin concentration constant. We then analyzed the recovered plasmid by digesting with XbaI and PstI and comparing the intensity of the digested plasmids on ethidium bromide stained agarose gel electrophoresis. The results below demonstrated that above a critical concentration needed to select for the double transformed cultures, copy number is unaffected by antibiotic concentration. [PICTURE]

Thursday, July 19

PCR the color constructs in order to biobrick them

Friday, July 20

Back to Weekly Log

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-We made plain LB plates and then plated 50 microliters of both wild type and the PAL mutant of <i>Synechocystis</i>.
 PCR the color constructs in order to biobrick them