Team:WashU/Week6

Digest To Check Construct

The minipreped constructs from Saturday were digested with XbaI and PstI to check if they contained the desired plasmid and insert.

A 1% agarose gel was made to check the digests of the ligation.

The ligations were deemed unsuccessful, and most were likely the plasmid backbone with the small self-ligated addition filling in the cut. We will re-digest the insert and plasmid and gel purify a large quantity of DNA to reduce the risk of self-ligation.

Redigest and Gel Run

Inorder to confirm that the ligation of Cs42s and psl2131 (a.k.a. Bert's plasmid) was not successful and that the all the colonies were just the plasmid religated with itself, we redigested the plasmid and analyzed it by agarose gel electrophoresis. The result was again a single band at a length consistent with the size of the plasmid.

Kan/C Plates

Kanamycin-chloramphenicol plates were made to select for E. coli that take up our construct to produce Saffron from Zeaxanthin and Part:BBa_K395704 which will bring E. coli to Zeaxanthin. Our construct will be Kan-resistant while Part:BBa_K395704 will be C-resistant. The plate will select for the double transformations. The plates were made using the LB-plate protocol on the protocol page.

Check Glycerol Stock

One of the glycerol stocks was checked by thawing a tube from Saturday on ice and adding .4 mL of it to 3 mL of LB+Amp liquid. The liquid will shake over night at 37°C. The rest of the glycerol stock was refrozen at -80°C.

YLC Color Constructs

Since the color constructs are proving difficult, and since we will not have the primers until Friday, we are rehydrating and transforming 4 others constructs to have the colors ready for the YLC project demonstration in one week. The following 4 parts were used to transform cells that will incubate at 37°C overnight:

RFP: I13521
GFP: I13522
YFP: 13604
CFP: S03475
75 mL of the LB solution that shook for an hour during transformation was used to plate the cells.

Tuesday, July 3

YLC Color Constructs

The plates all have a good amount of colonies on them.

The RFP and GFP are glowing well after about 14 hours of incubation. They are now sitting and room temperature while the CFP and YFP plates are placed back in the incubator to further develop.
The GFP and RFP plates both had colonies picked from them to prepare in Amp liquid culture. These will be used to create sample plates for the YLC and start preparing solutions with which the campers at the YLC will BioPaint.

Saffron Constructs Preparation

Today, the Kan-plasmid which we are using as the construct vector and the CS42s construct to produce Saffron from Zeaxanthin were both digested with XbaI and SpeI. These digests were then run on a gel to prepare for gel purification. The gel from yesterday was not a clear gel result so this gel was a .35% gel to try to further see separation of bands.
In the gel below, P refers to the plasmid PSL2131, and C and CIII refer to two different colonies of our CS42S construct.

Afterwards, gel purification was done on the plasmid and constructs. However, due to some error on our part, no DNA was recovered, as shown by the nanodrop.

Glycerol Stock Check

The stock that was grown in LB+Amp overnight was cloudy suggesting cell growth. A glycerol stock that was made on Saturday of a GFP will be checked today by plating it on an LB+Amp plate. This test will be more conclusive in showing the ability of the glycerol stock to not only produce growing bacteria but also maintain plasmid at -80°C.

Thursday, July 5

YLC

In preparation for our trip to the Youth Learning Center next Monday, the team prepared large cultures of red and green fluorescent protein constructs. The YFP and CFP constructs both fluoresce only faintly and thus have been discarded for the purposes of our YLC visit.

Saffron in a Kan

We received our primers today, and performed a PCR.

Friday, July 6

PCR

We ran a gel on the rest of yesterday's PCR. The gel, shown below, suggests the presence of primer dimers indicating that our PCR failed. After troubleshooting, we will attempt to get the colony PCR to work anew. (In the gel, A and K refer to Ampicillin and Kanamycin plasmids, Z refers to the ZCD gene, U refers to UGTCS2, and C refers to CrtZ.)

Troubleshoot and redo failed PCRs

6803

Because our first attempt at transforming 6803 with a kanamycin resistant vector did not succeed due to the plates dying out, we attempted to carry out the procedure again using our new incubator. We plated a total of four BG-11 plates, giving us the ability to do a 2x2 test of plate conditions. We chose to test the effect of mixotrophic media (with and without glucose) and amount of transformed cells plated (50ul vs 200ul) on the transformation efficacy.

Saturday, July 8

First, we reinnoculated the ligation cultures.
Then, we maxipreped PsbA2 and used the nanodrop to see how much DNA resulted from the prep.

Next, we digested the ligations, miniprepped and digested them, made a gel, and ran the gel. [PICTURE]

In addition, we started a 20 degree culture of E. coli with the Z construct and our construct CS42S.

We miniprepped the E. coliwhich had taken up double plasmids (Z and C plasmids). We digested these and then ran them on a gel. [PICTURE]

Finally, we replated ligation 2, our most successful ligation of plasmid PSL2131 and construct CS42S.