Team:WashU/Week8

Monday, July 16

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

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 three colony PCRs, using colonies 2, 4, and 13. However, the PCRs were all unsuccessful and resulted only in primer dimers.

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.

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
We discovered that our problem with the PCRs was due to not optimizing the amount of magnesium chloride needed for the Taq polymerase. Instead, we had been using the Taq buffer with magnesium chloride already added, which was not suited for the reactions we were running. In the gel picture below, the first three wells contain DNA using the buffer with pre-added magnesium chloride, and the last three wells show what happens when we calculate and manually add the proper amount of MgCl2 to optimize Taq polymerase activity. (Also, Z=ZCD, U=GTCs2, and C=CrtZ.)

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. (2x refers to ligation cut with both X and P, 1x indicates ligation cut with P, and 0x signifies uncut PC42) The band sizes resulting from the digestion match the sizes of the plasmid and construct, indicating that the ligation worked.

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 chloramphenicol 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. In the picture below, the wells, from left to right, have 5 microliters of DNA followed by 10 microliters of DNA per each concentration of chloramphenicol, which is labelled on the graph as 0, 25, 50, 75 and 100 mM.

Thursday, July 19

To determine whether our culture of Synechocystis is contaminated or not, we plated 50 microliters of both the PAL mutant and wild type Synechocystis on plain LB agar plates.

In order to prepare some of our constructs for sequencing, we ran PCRs for the color (1=YFP, 2=GFP, 3=RFP, 4=CFP and 8=mCherry) and our Z, U and C constructs. The products were run on a gel, displayed below.

Friday, July 20

After using a PCR cleanup kit to purify our results from yesterday, we then digested the constructs and ligated them into plasmids, and plated the final products.