Team:KU Leuven/Journal/MeS/wetlab

This week we transformed DH5α cells with the methylsalicylate producing brick from MIT 2006 (Bba_J45700). Since this brick has got two antibiotic resistance genes (kanamycin and ampicillin), we plated them out on kanamycin plates and ampicillin plates.
Using the cells containing Bba_J45700, some of our team members did a smell test, to see whether the production of methylsalicylate was significant, since methylsalicylate has a wintergreen scent. Plate C was the one containing these cells. These are the results, “-“ meaning no difference in scent compared to the control, “+” meaning a difference in scent compared to the control. As can be seen, plate C is chosen only a few times. The brick might need some improvement, for example by using stronger promoters and ribosome binding sites. So we will reconstruct Bba_J45700 by taking out the useful pieces and putting them back together with other parts.

Name	plate A	plate B	plate C
Ingmar	+	-	-
Su	+	-	-
Sander	+	-	-
Bert	-	-	-
Frederik	+	+	+
Saar	-	-	+
Robbert	+	-	-
Sabine	+	-	+
Flore	+	+	-

In order to get pchBA and BSMT1 out of the original brick, a PCR on pchBA and BSMT1 is done. The fragments are cut with EcoRI and PstI, ligated in pSB1C3 and transformed.

Last week’s transformation failed, so we did PCR purification to make sure that all the small fragments were gone. The transformation was repeated.
To improve the production of methylsalicylate, we wanted to improve the production of chorismate, a precursor of methylsalicylate. The product of the aroG gene is an important enzyme in the production of chorismate. We first wanted to isolate this aroG, and then mutate it to make it insensitive to negative feedback from phenylalanine.
We did a colony PCR on 4 random DH5α, to amplify aroG.
Fragments were all around 1000 bp, as expected.
Restriction with EcoRI and PstI, ligation and transformation.

Our transformations for aroG, BSMT1 and pchBA were all succesful. To check whether the genes were actually inserted into the plasmids, we did a colony PCR on all three (in five-fold, five colonies of each gene).
Unfortunately, only the AroG band had the right length. For the other two genes, only very small fragments were visible. We inoculated the aroG containing cells.
For the mutagenesis of aroG, we performed a mutagenesis PCR with primers with a point mutation in them. After this, we did a KLD treatment (kinase, ligase, DpnI) + transformation.

Mutagenesis didn’t work since there was no PCR product to be seen on a gel. We performed the mutagenesis with a high fidelity polymerase in a gradient PCR with different annealing temperatures (53-55-58-61-64°C) in parallel.

On a gel, we put:

• lane 2: the original plasmid containing AroG
• lane 3: the original plasmid containing AroG cut with EcoRI to make it linear
• lanes 4-8: the PCR products of the gradient PCR, one for each annealing temperature

After visualisation, there were PCR products visible with lengths corresponding to the length of the cut plasmid, as expected.
After ligation and transformation, there were no colonies to be seen, so we repeated this with an extra DpnI treatment to make sure that all the original plasmid was cut into smaller fragments that wouldn’t be able to be transformed. Unfortunately, also this transformation was unsuccesful.