Team:KU Leuven/Journal/EBF/wetlab

Some of us have already finished their exams so we started working in the lab, preparing everything for the main work that wil start July 1st. We poured the agar plates with and without the antibiotics and made the FSB buffer that will be needed to make competent cells.

Finally, all of us are finished with the exams, so we can really start working in the lab, We started made chemically competent Top10 and DH5alpha cells. To test our competent cells we tried the iGEM transformation efficiency kit, but surprisingly no colony appeared in any of them! Now we have to figure out what went wrong. Possible reasons are:

• Bad competent cells
• We didn't use enough recovery time for the cells.
• The cells need more time to grow.

To examine what went wrong we did the experiment over again, but this time we used our own pUC 19-vector and let the cells recover for 2 hours instead of one. Now we do have cell growth, so we counted the cells and calculated the transform efficiency. This was still quite low though...
We prepared the GTE-buffer for future use and prepared agar medium. Later, we found out that the medium in the autoclave spilled out everywhere in the autoclave, possibly due to the pressure. Lukas was the lucky guy who got to clean out the autoclave.
The parts from iGEM have arrived! We selected 16 parts to work with further to make our EBF-producing-devices.

These parts were transformed to our chemically competent cells. During the heat shock the heat automatically turned off after one round, wich we didnt realize at first. So for bricks 1I, 7E, 5E, 20O, the heat shock temperature is a bit lower (around 37°C). Hence during the plating we used 50µl of transformed competent cells instead of 20µl for these samples. The cells were left to grow over the weekend in medium containing chloramphenicol.

Unpatient as we were, we first checked the plates of the transformed competent cells and found out that only 4 petri dishes have a small amount of colonies (7E2 top10, 5E3 top10, 1G1top10, 5E3 DH5alpha). Interestingly, the ones where we used 50µl of cells to plate instead of 20µl, were most succesfull. This means that the lack of colony may be caused by a too low amount of cells put on the petri dishes. This in turn also suggests that the transforming efficiency is quite low. We repeated the transformation with 50µl or even more cells for the unsuccessful biobricks. Later we also performed the plasmid extraction on all the successfully transformed biobricks.
We wanted to do a colony PCR on Streptomyces coelicor, so first we prepared the primer stocks for that. Streptomyces have a gene coding for bèta-farnesene synthase. This enzyme produces EBF out of farnesyl diphosphate.
We extracted the genome from Streptomyces using 4 different methods (as Streptomyces is gram negative, it is a bit difficult to extract the genome):

• Microwave Streptomyces for 4 mins
• Streptomyces in water with 0.2%SDS, 4 min microwave.
• Streptomyces in water and 1% SDS, 4 min microwave.
• Streptomyces in TE buffer, 0.2% SDS, 4 min microwave.

We ran a colony PCR for all these Streptomyces and a negative control. We cleaned up the PCR product and ran the gel of the DNA we got from the colony PCR. The gel was stored in TAE buffer at 4° C, so that we could visualize the gel next Monday.
We also tried to make competent TOP10 and DH5alpha cells using the Inoue method instead of our regular chemical competent cells.

Amplify Backbone

As the amount of backbone DNA we received from iGEM is very small, we decided to amplify the backbones ourselves. For this we chose 3D1, a small biobrick coding for a double terminator. We inocul the 3D1 transformed strain into 3 ml LB medium, then isolated the plasmid and cut the backbone out of the plasmid. To make sure that we have enough backbone we did this in quadruple. Before we did the digestion, we ran a nanodrop for these 4 samples to check the DNA concentration.

The digestion was done with EcoRI+PstI.

The band of the backbone does not fit our expected band very well so we decided not to use these backbones.

Device

We visualized the gel we ran last Friday, containing the EBF-producing genes that were obtained from the colony PCR. For pretreatment 1 and 2 we can see a clear band of DNA. For these we than ran a high fidelity PCR. These PCR products were made into stocks and diluted 15 times (1µl PCR products + 14µl miliQ). Then we purified the PCR products and did a nanodrop for the PCR products we got:

To make our very first biobrick we performed digestion and ligation of the EBF1 gene and the chloramphenicol backbone that we received from iGEM.

Biobricks

We also performed plasmid isolation for the successfully electroporated biobricks (1I2, 1G1, 5E3, 2M5, 1H5, 7E2).
We ran nanodrop for the biobrick plasmids we want to digest.

Then we digest these parts with EcoR1+SpeI, except for 17D1 which was digested with XbaI+PstI. For all of them we used 1µg of DNA except for 17D1 (434.4ng due to the limited amount we have). We ran a gel with 20 µL of the product. The rest of the digested DNA we stored at -20°C after denaturing the enzyme by putting it at 80 °C for 20min.
After visualizing the gel with EtBr, we can only observe the bands of the backbone (and some other bands that we don’t know where they come from), the band of the parts we want cannot be seen. The possible reason could be that these parts are too small (they are promoters, RBS, etc, with a size rangeing from 12 to 72bp), hence the EtBr cannot bind them in a sufficient amount and therefore we cannot see them. This is why we will need another strategy to tackle the problem of assembly.
We transformed Inoue competent cells with 4F3 5D3 23L5 (an alternative for double terminator) 5E1 and EBF. All these transformations were successful! We inoculated these cells with 4ml LB with correct antibiotics, incubated them at 37°C over night and transfered them to 4°C.

Device 2

First we digested the Ampicillin, Tetracyclin and Kanamycin backbones from iGEM with EcoRI+PstI restriction enzymes and purified the DNA with the PCR purification kit. We digested EBF1 with Xba1+PstI restriction sites, purified it with the PCR purification kit, then ligated the digested 3O1, EBF1 and Kan backbone to make our second device. We left the incubation of the ligation at room temperature over the weekend.

We extracted the plasmid from the cultivated cells containing 5E1 4F3 23L5 and D1 (device 1) with the use of the plasmid purification kit, and ran the nanodrop for the isolated plasmids:

In the afternoon, we digested 5E1a with EcoRI + SpeI and D1,1 and D1,2 with EcoRI + PstI. We ran a gel for D1,1 and D1,2 to confirm we have the E-beta-farnesene synthase gene. Then we ligated 5E1 + EBF (=D1)+ pSB1K3 (the standard iGEM backbone). We incubated it at room temperature overnight.
We ran a gel for these, but the results were not exactly cheering. We only got 1 band from the digested EBF device D1, the possible reason could be that the backbones ligated to eachother to form a bigger plasmid, instead of inserting the EBF into the plasmid. Since we only took 2 colonies, these results are possible.
We did a colony PCR for 10 randomly selected D1 colonies and ran the gel to confirm they have D1 plasmid. The results are weird however, we only got a band with a short length of DNA. A possible reason could be that the anealing temp for the primer was a little low so that the primer anealed unspecifically, which lead to the short secquence products.
Therefore we randomly selected 17 more colonies, plus 10 colonies from yesterday, and performed another round of colony PCR. The nanodrop results for these colony PCR are really high, meaning that the PCR worked. We later ran the gel, but the results were again not pleasant. It's very possible that the ligation (or even digestion) was not good.
We transformed D2 and D3 (D2 is promoter 3O1 in front of EBF, D3 is promoter 5E1 in front of EBF) into inoue cells. This wass unsuccessful so we tried again with electroporation. We transformed D2 and D3 by electroporation and plated out the transformed cells on Kan plates. The tranformation was again not successful, so we tried again with a higher DNA concentration.
The EBF-plasmid finally arrived. This plasmid is the (E)-beta-farnesene synthase from Artemisia Annua in the pET28 backbone. The plasmid was on filter paper, so we used an elution buffer to retrieve it. We transformed the EBF plasmid to competent cells from the mutagenesis kit (with this kit we want to mutate a specific site in the EBFaa pET28 plasmid)) using electroporation and plated them out on Kan plates. This transformation was successful.
We started our new strategy for digestion and ligation: First we ran a nanodrop for the bricks

Then we did digestion for the chosen bricks, this time we digested 2µg of DNA instead of 500ng. the digestion sites:

We incubated these @ 37°C for 25-30mins and inactivated @ 80°C for 20mins. Then we did a PCR purification for the digested bricks as well as the chloramphenicol backbones. Finally, we ran a nanodrop. The results, hower, are really weird, indicating there is barely any DNA. This means that the reason that the transformations of D2 and D3 all failed this week is because we dont have any backbones to ligate!
The problems happens at the purification step!