Today, we prepared LB Agar to autoclave in order to prepare the plates that will be needed soon for our bacterial cloning. Then, we prepared the plates with kanamycin and ampicillin, because the two plasmids required for the cloning protocol have the respective antibiotic resistance genes.
Today we received the gBlocks that we had ordered to IDT on 19th of August. The first lab task was to resuspend the two gBlocks provided by IDT in nuclease-free water, according to IDT’s gBlock protocol. These two ordered gene fragments were:
Tryptophan decarboxylase gene (TDC)
Tryptophan hydroxylase gene (TPH)
The plates with E.coli transformed with the plasmid pET15-MHL were generously lent by the “Protection of cultures” investigation group of the Public University of Navarre (UPNA). Then, we prepared LB with ampicillin in order to inoculate a liquid bacterial culture. The overnight incubation at 37ºC was indispensable.
The plasmid extraction or “miniprep” was performed following the instructions of a DNA extraction kit. This way we obtained our plasmids of interest which will be used as cloning vectors for the TPH cloning. After that, they were checked by using a gel electrophoresis.
First, we performed a PCR to add the endings required to the LIC cloning method to the TPH gBlock. Then, we purified the PCR products by diagnostic gel electrophoresis. Meanwhile, we digested the vector pET15-MHL with the type IIS restriction enzyme BseRI with the aim of linearizing it. We also prepared more agar plates with kamamycin and others with ampicillin. It is essential to perform the whole procedure in a sterile environment to avoid contamination.
Today, the overhangs of the plasmid and the insert were created thanks to the 3’ exonuclease activity of the T4 DNA polymerase. On the one hand, the linearized vector was treated with T4 DNA polymerase to "chew back" the free 3' ends, in the presence of dGTP as the only free nucleotide in the reaction. Simultaneously, the insert was submitted to the same reaction with the difference that instead of dGTPs, there were dCTPs. These two reactions were incubated at room temperature for 30 minutes and then, inactivated by heating to 75° for 20 minutes.
Then, the two reactions’ products were mixed together and the annealing step took place. In the afternoon, we transformed TOP10 bacteria with the final product by heat shock.
This afternoon, we realized that no colonies had grown. So we focused on optimizing the digestion and ligation protocols after a time of reflection in the group. We decided to start the cloning experiment of the second enzyme because it was based on simpler and more standardized protocols.
Today, we started the first step of the cloning experiment of TDC using pET28a. The plates with E.coli transformed with the plasmid pET28a were generously lent by the “Protection of cultures” investigation group of the Public University of Navarre (UPNA). Then, we prepared LB with kanamycin in order to inoculate a liquid bacterial culture. The overnight incubation at 37ºC was indispensable.
The plasmid extraction or “miniprep” was performed following the instructions of a DNA extraction kit. This way we obtained our plasmids of interest which will be used as cloning vectors for the TDC cloning. We were in the lab until the university closed, so we enjoyed the beautiful dusk!
Today we had a group meeting to organize the tasks of the following weeks.
We performed an electrophoresis to ensure the presence of the plasmid. However, we realized that it was not the band pattern we had expected previously because circular DNA migrates differently in gel electrophoresis.
We performed the digestion of pET28a with HindIII and EcoRI and we left it for incubation overnight at 37ºC, because both of the restriction enzymes own the same optimal temperature. On top of that, we performed a PCR to include the restriction sites of HindIII and EcoRI flanking the TDC gene in order to be able to insert it inside the digested plasmid.
We ran the diagnostic gel of the digested vector and we visualized it using a ChemiDoc. However, we purified our bands of interest, which were those of 5350 bp. A UV transiluminator was required to cut the bands.
In addition, we inoculated a colony containing pET15-MHL, which will be required for the second construct.
Today, we extracted the other plasmid of interest, pET15-MHL. Furthermore, we digested the insert (TDC) with the corresponding enzymes in order to get the sticky ends to develop the ligation. When we measured the concentration of the DNA in the Nanodrop, it was so low that we realized that the purification must be performed with the minimum number of lanes. We used four for the same DNA of interest!
We were so eager to work that we went to university on Saturday! We were the only students in the whole campus… First of all, we inactivated the enzymes. It was required a 20 minutes incubation at 65ºC and then another one but at 80ºC. The reason was that EcoRI must be inactivated at 65ºC and HindIII, at 80ºC. After that, the comprobation gel electrophoresis was performed in the ChemiDoc. The bans had a very low intensity due to the low concentration.
We ligated the digested insert and vector using an ultra rapid DNA ligation kit. We also decided to put the wide vector (without insert) as a positive control of religation, in order to compare the plates and see the background. Then, we transformed TOP10 bacteria by heat shock and we plated them in agar plates with the antibiotic. We added two different volumes per condition to facilitate the following inoculation. We also prepared more agar plates with kanamycin in the case the transformation did not work and it would be repeated.
We got up early to discover if the transformation had been successful. Unfortunately, we found out that just a few colonies had grown up. We inoculated them in liquid medium. However, we did not trust the existence of well transformed colonies so we decided to repeat the transformation on Wednesday but trying to improve the different conditions.
Unluckily, as there was no more growth on the plates, we performed the transformation again.
This time, we used two methods for bacteria transformation: heat shock and electroporation. We also selected three bacterial strains to improve the probabilities of success. So we developed the electroporation with the electrocompetent strain: BH5⍺ and the heat shock with the chemically competent strains: TOP10 and JM109. We also plated two volumes per condition (including the three controls). So, in conclusion, we had 12 plates with transformed bacteria! We left them for overnight incubation at 37ºC.
Unfortunately, the obtained colonies showed bizarre morphology. The electroporated ones (DH5𝝰) were considerably smaller than expected. The two strains of E.coli transformed by heat shock (TOP10 and JM109) did grow, but in a reduced number of colonies. So we decided to repeat all the cloning steps from the beginning.
We performed again the digestion of both the insert and the vector with EcoRI and HindIII, we ligated them and we transformed bacteria by electroporation and heat shock. We left them incubating overnight.
In the morning, we could appreciate the growth of colonies in the electroporated bacteria plates. We inoculated 8 colonies of DH5𝛂 and 3 colonies from the control condition of DH5𝛂. What surprised us the most was the fact that more colonies grew in the transformation with the insert than in the control condition.
Today was the last day in the lab. We did minipreps from the 8 colonies incubated overnight at 37ºC and we measured their concentrations using the Nanodrop:
| Sample | Concentration (ng/µL) | Sample | Concentration (ng/µL) |
|---|---|---|---|
| 1 | 330.0 | 5 | 561.2 |
| 2 | 287.8 | 6 | 254.1 |
| 3 | 49.7 | 7 | 732.2 |
| 4 | 66.3 | 8 | 390.2 |
Then, we digested them with the two enzymes and we checked the presence of the insert by gel electrophoresis.
As the first cloning experiment (TPH on pET15-MHL using LIC) did not work out, we must repeat the steps in order to get synthetic bacteria with the recombinant plasmid. Once we have the two types of transformed bacteria, we must purify the enzymes by affinity purification on columns based on their histidine tags. After that, the tryptophan extracted from by-products will be added to the in vitro reaction that includes both enzymes to get the serotonin. Finally, the serotonin will be extracted by using HPLC and it will be nanoencapsulated by a local biotech startup.