Team:Tec-Chihuahua/Results
While we were constructing our BioBricks™, we realized there was a problem because the insert wasn't ligating to pSB1C3. Everything seemed correct in the transformation protocol, but once the DNA was visualized on an agarose gel our insert was missing and only pSB1C3 was evident. We then thought there was a problem during ligation. Therefore, we analyzed IDT’s synthesized sequence and identified that there were missing additional base pairs that flanked the restriction site so that the enzyme could properly cling and cut the sequence. This issue was troubleshot with the TecCEM iGEM team. They had the great idea to create primers that add six base pairs before the prefix and after the suffix thereupon amplifying the gene of interest. This collaboration was crucial for the construction of our BioBricks. Read the entire collaboration.
BioBrick assembly
The three composites of our creation, apidaecin - [BBa_K2834003], defensin 1 - [BBa_K2834005], and abaecin - [BBa_K2834006] were characterized with the intention of expressing the peptides in E. coli BL21 (DE3) by IPTG induction. Its antimicrobial activity was evaluated against Gram-positive bacteria with antibiotic susceptibility testing by measuring OD600 in broth.
The three composites were synthesized by IDT® with the prefix and suffix flanking the region of interest. The final parts resulted in a sequence of 310 bp for apidaecin, 415 bp for defensin 1, and 361 bp for abaecin. Once the synthesis arrived, and the PCR was carried out, double digestion with EcoRI-HF and PstI restriction enzymes was made to each composite, and the chloramphenicol linearized plasmid backbone (pSB1C3) for following ligation of the backbone with each one of the fragments. This resulted in complete expression plasmids of 2337 bp for apidaecin, 2442 bp for defensin 1, and 2388 bp for abaecin. Afterward, Escherichia coli BL21(DE3) cultures were transformed by heat shock for following antibiotic selection of clones. Next step consisted of plasmid extraction and electrophoresis of the undigested plasmids, linearized plasmids with one enzyme, and double digested plasmids. Agarose gels allowed the confirmation of the correct plasmid constructions.
Protein production
IPTG induction and extraction
Following the construction of each BioBrick, it was necessary to induce protein production. Since the T7 promoter regulates transcription of the construct, isopropyl β-D-1 thiogalactopyranoside (IPTG) is used as an inducer for T7 RNA polymerase production. The concentration of IPTG used was 0.5 mM. After induction, the cultures were incubated for six hours at 37 °C and 225 rpm. After that, protein extraction by lysis solution was made in order to obtain the soluble peptides. For insoluble peptides, the sample was treated with lysis solution + 6 M urea.
SDS-PAGE
After protein extraction, electrophoresis in polyacrylamide gel (12%) was performed to corroborate the peptides of interest were indeed expressed. To calculate the molecular weight of each peptide, the Promega Biomath Calculator was used.
Bands of the following kDa were expected:
- Abaecin - 4.62 kDa
- Apidaecin -2.75 kDa
- Defensin 1 - 5.94 kDa
Antimicrobial susceptibility testing
In order to prove the antimicrobial activity of abaecin, apidaecin and defensin 1, antimicrobial susceptibility tests were performed for two different bacteria: Bacillus subtilis and Streptococcus pyogenes.B. subtilis was chosen because it is one of the best known Gram-positive microorganisms and S. pyogenes was chosen because it is one of the most important bacterial pathogens to humans. They both are widely known, commonly used, and thus allowed to better analyze the activity that the peptide has.
Being unable to isolate our peptides by affinity tag purification due to lack of equipment, crude protein extract was used in the experiment. In order to validate the experiment, different concentrations of the peptides and several controls were used; 12 ml of LB broth, with a bactericide agent or a control, were inoculated with 100 μL of the overnight culture of each bacteria. Afterward, OD600 was measured at 3, 6, 9, and 21 hours after inoculation.
Defensin 1
B. subtilis (figure 3a) was treated with 30.6 μg/mL (LC) and 153 μg/mL (HC) of total proteins of transformed E. coli BL21 (DE3) with defensin 1. Also, it was treated with 29.565 μg/mL (LC) and 147.825 μg/mL (HC) of untransformed E. coli BL21 (DE3) for negative control. A culture of B. subtillis was used as a negative control as well. At 21 h, both concentrations of defensin 1 produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with defensin 1 OD600 decreases in 14.23% and with the highest one it decreases in 16.28%.
S. pyogenes (figure 3b) was treated with the same concentrations as B. subtillis. At 21 h, both concentrations of defensin 1 produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with defensin 1 OD600 decreases in 8.26% and with the highest one it decreases in 12.99%.
Figure 5. Antimicrobial susceptibility testing results for defensin 1. a) B. subtilis challenged with low (LC) and high (HC) concentrations of total proteins of transformed E. coli BL21 (DE3) with defensin 1 and total proteins of untransformed E. coli BL21 (DE3). b) S. pyogenes challenged with low (LC) and high (HC) concentrations of total proteins of transformed E. coli BL21 (DE3) with defensin 1 and total proteins of untransformed E. coli BL21 (DE3).
Apidaecin
B. subtilis (figure 2a) was treated with 28.52 μg/mL (LC) and 142.6 μg/mL (HC) of total proteins of transformed E. coli BL21 (DE3) with apidaecin. Also, it was treated with 29.565 μg/mL (LC) and 147.825 μg/mL (HC) of untransformed E. coli BL21 (DE3) for negative control. A culture of B. subtilis was used as a negative control as well. At 21 h, both concentrations of apidaecin produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with apidaecin OD600 decreases in 15.15% and with the highest one it decreases in 15.75%.
S. pyogenes (figure 2b) was treated with the same concentrations as B. subtilis. At 21 h, both concentrations of apidaecin produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with apidaecin OD600 decreases in 10.2% and with the highest one it decreases in 12.45%.
Abaecin
B. subtilis (figure 2a) was treated with 29.74 μg/mL (LC) and 148.7 μg/mL (HC) of total proteins of transformed E. coli BL21 (DE3) with abaecin. Also, it was treated with 29.565 μg/mL (LC) and 147.825 μg/mL (HC) of untransformed E. coli BL21 (DE3) for negative control. A culture of B. subtilis was used as a negative control as well. At 21 h, both concentrations of abaecin produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with abaecin OD600 decreases in 16.08% and with the highest one it decreases in 17.16%.
S. pyogenes (figure 2b) was treated with the same concentrations as B. subtilis. At 21 h, both concentrations of abaecin produced a decrease in OD600 compared to the untransformed E. coli BL21 (DE3) control. With the lowest concentration of total proteins with abaecin OD600 decreases in 3.77% and with the highest one it decreases in 14.39%.
Successes & Failures
The iGEM experience taught us a lot of unique things, and as we had significant achievements, we went through many unexpected obstacles. Throughout the year we faced many challenges for finally reaching our goal; we believe that the effort was worthwhile.
Model
Successful
With minimal mathematical and informatics background, we were able to adapt a pharmacokinetic model to our situation and code effective solutions for diffusion data analysis. This model allows us to approximate peptide concentrations within a nanocapsule at several time intervals; it can also help further developments in similar projects.
Unsuccessful
Our mathematical model went through many iterations (no pun intended) before arriving at its final state: we tried to develop a molecular model to evaluate protein production and a populational model to predict the health of the beehive by eliminating nodes of infection. In the end, time and resources forced us to concentrate our efforts on the most pressing of matters: peptide delivery.
We were unsuccessful in solving the equation for D; we tried several series properties and other sorts of tricks, but we couldn’t find an analytical solution. We decided to find the diffusion coefficient with a simulation of multiple lines.
Our most poignant failure was not being able to run the experiments successfully. Apidaecin was retained at a customs office for a whole month. By the time we needed the nanoencapsulation to be at the lab for experimentation, the peptide had not yet arrived. We are left with a burning vigor to help make importation easier for all sorts of experimentation in Mexico.
Laboratory
Successful
We are the first iGEM team from Tec-Chihuahua that manages to send successful results for the InterLab Study. Besides being a rewarding experience, it was a lot of learning since we had never used a microplate reader.
Unsuccessful
At the beginning, we worked with E. coli TOP10 but later we had to change to DH5a since the TOP10 strain was contaminated.
For a few weeks, the successful transformation of our competent cells was difficult. We discovered that the key was in the quality of the competent cells. We tried to make several stocks of competent cells with glycerol and store them at -20 °C, however, this did not work. We had to make the competent cells on the same day that the transformation protocol was to be carried out.
We worked in the transformation of competent cells of DH5a with the synthesis of our part linked with pSB1C3. When they were disseminated in the LB medium with chloramphenicol, there was a presence of colonies which meant that they had been correctly transformed. However, when performing the electrophoresis of plasmid extraction only one band of the size of pSB1C3 was observed. First, we thought that the protocols of digestion and ligation that we carried out were not the correct ones or there was a bad manipulation. This idea was discarded when using a control with RFP. Doing more research, we discovered that the parts that we sent to synthesize in IDT lacked base pairs next to the restriction sites of EcoRI-HF and PstI. This means that the enzyme has no way to attach to the DNA sequence and digest it. Why did we have colonies that grew on LB + CAM? We believe that the re-circularization of the plasmid was possible.
Future Plans
Eight months are not enough to address the full scope of our project. This does not end here!
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