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 aroG 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.

We decided to do something different to try and mutate aroG, by making us of a megaprimer. This megaprimer was made with a regular primer, starting from the biobrick suffix, and a primer containing a point mutation.
In order to get rid of the primers that were used in the PCR reaction of this megaprimer, we put the product on a gel containing crystal violet. The purified megaprimer was used in a PCR reaction with the aroG containing plasmid, in order to mutate it.

35µl	H2O
10µl	buffer
0.5µl	dNTP
2µl	megaprimer
1.5µl	aroG plasmid
1µl	Phire polymerase

For the mutagenesis of aroG, we ran a PCR reaction without polymerase, to have a comparison on the gel.
No bands were visible for either the positive and the negative mutagenesis PCR reaction. However, it is possible that a cluster of annealed plasmids didn't move into the gel, since there is a high signal in the well. Luckily, Misha told us, E.coli is capable of untangling this, so a transformation might still work.
We also put the methylsalicylate producing brick into a new backbone, pSB1C3.
In order to reconstruct the methylsalicylate brick for higher production, we also transformed the bricks 13B5 and 1M3, respectively pTetR+RBS and the lacI coding sequence on ampicillin and chloramphenicol plates, respectively.

We started the construction of new biobricks:

• pTetR + strong RBS
• lacI + strong RBS
• strong RBS + MIT 2006 brick

The methylsalicylate producing brick (MIT 2006) in the pSB1C3 backbone was cut with EcoRI and PstI and put on gel. (Picture below, second lane, two bands: approximately 2000 and 3000 bp) We will now send this brick to be sequenced, so we will get a confirmation that this is indeed an improved brick.
For the mutation of aroG, we repeated the mutagenesis PCR with the megaprimers,one reaction with polymerase (+) and one without (-).
One part of this was put on gel (picture below).

• lane 3: (-)
• lane 4: (+)
• lane 5: megaprimer
• lane 6: aroG containing plasmid


The rest was DpnI treated, and dialysed to remove salts, by adding it to a 2% agarose + water gel.
After 30 minutes the contents of the well were pipetted out and electroporated.
Furthermore we performed a Phire II PCR reaction on our methyl salicylate brick, to obtain good PCR products of the bsmt1 and pchBA genes seperately (we need to get rid of the lacI operon in between, since it would interfere with the rest of the model).
Our reaction composition was as follows:

10 µl	5X Phire Reaction Buffer
1,5 µl	Forward primer
1,5 µl	Reverse primer
1 µl	dNTPs (10mM)
1 µl	Template DNA
1 µl	Phire II Polymerase
34 µl	Milli-Q water

The PCR program we used was:

98°C	30s
98°C	5s |
X °C	5s | x35
72°C	Ys |
72°C	60s
4°C	hold

• X = 70°C for bsmt1, 73°C for pchBA
• Y = 20s for bsmt1, 50s for pchBA

This week we performed different digestions and ligations to obtain several constructs for our final plasmids.:

1. aroGM with E and S
2. TT with E and X
3. pTetR + RBS with E and S
4. lacI + RBS with E and X
5. pchBA PCR product with E and P
6. pchBA PCR product with E and S
7. bsmt1 PCR product with E and P
8. bsmt1 PCR product with E and S

After digestion we performed 6 different ligations:

1. aroGM (E+S) with TT (E+X)
2. pTetR+RBS (E+S) with lacI+RBS (E+X) in a
3. pchBA (E+P) with Ampicillin backbone (E+P)
4. bsmt1 (E+P) with Ampicillin backbone (E+P)
5. bsmt1 (E+S) with TT (E+X)
6. pchBA (E+S) with TT (E+X)
After ligation, we performed chemical transformation of these ligation products in Inoue treated competent cells (TOP10). For 1, 5 and 6 we use plates containing chloramphenicol, for 2, 3 and 4 we use plates containing ampicillin.

Unfortunately, after extracting these plasmids and cutting them with EcoRI and PstI, we didn’t see the expected bands. We repeated the digestions and ligations of the previous constructs, but without succes.
This week, we started working on designing the gBlocks we were going to order. For the construct with pTetR-RBS-lacI-RBS-aroGM-TT we used 4 gBlocks, for the improvement of Bba-J45700, we used just one that contained a ptetR sequence in stead of a lac-promoter that would be affected by the lac repressor. The rest of the brick (the two parts to the side of this gBlock) was made with PCR of the original brick.

We inoculated the transformed expression strains (BE21) for the use of making growth curve and protein extraction. The growth curve experiment was performed under 37degrees, the samples order was:

1. gEBFaa5
2. HD12
3. HD13
4. gEBFaa5 + 5mM Mg2+
5. HD12 + 5mM Mg2+
6. HD13 + 5mM Mg2
7. control: BL21 strain

Meanwhile, the protein expression was performed in different conditions as well:
At 37°C:

1. MS + 0.2mM IPTG (induced with IPTG again at OD of 1.89)
2. MS + 0.2mM IPTG + 1mM salicylate (induced with IPTG again at OD of 1.47)
3. MS + 0.2mM IPTG + 1mM chorismate (induced with IPTG again at OD of 1.83)

At25°C:
4. MS + 0.2mM IPTG (induced with IPTG again at OD of 1.91)
5. MS + 0.2mM IPTG + 1mM salicylate (induced with IPTG again at OD of 1.67)
6. MS + 0.2mM IPTG + 1mM chorismate (induced with IPTG again at OD of 1.84)

With the extracted proteins, we ran the SDS-PAGE.

Afterwards, we prepared 68 epps to perform a double blind smell test of our methyl salicylate brick (BBa_K1060003) and the methyl salicylate brick from MIT 2006 (BBa_J45700). In this experiment 4 sets were used with termperature and incubation time as variables. The first set was conducted at room temperature for 24 hours, the second set was incubated at 37ºC for 24 hours, the third was incubated in 37ºC for 8 hours, the last one was carried out at room temperature for 8 hours. In each set, we inoculated our brick BBa_K1060003 with IPTG, IPTG + salicylate (concentration ranges from 0.01mM to 1mM), and IPTG + chorismate (concentration ranges from 0.01mM to 1mM), the same was done for the MIT 2006 brick BBa_J45700. These combinations of additives were also introduced in parallel for a negative control (non-MS producing E.Coli).

We had 11 people participating in the blind test. After the test we calculated the percentage of people who smelled MS. Based on the percentage, we can conclude that the smell was stronger after 24 hours incubation time compared to 8 hours. Incubating the bacteria in 37 degrees enhanced the production compared to incubation at room temperature. Another effect is a more obvious smell in the epps where 0.1mM or 1mM salicylate was added. In addition, the effect of adding chorismate became clearer after 24 hours. The reason why chorismate requires more time to see the effect might lie in the fact that chorismate is the very first precursor of the methyl salicylate production pathway, whereas salyciate is a precursor one step before methyl salicylate.
Most importantly we can conclude that our brick worked in terms of producing methyl salicylate.