The purpose of the experimental work was to express the DrsB1 and PcOSM constructs in the selected chassis E. coli BL21 (DE3), in order to produce these recombinant antimicrobial peptides with the objective of developing a fungicidal solution. This section describes the experimental work performed during the past months. An explanation of the assembly of the registered constructs and the strains we used for transformation to obtain the antimicrobial peptides of interest are shown. Additionally, the expression of mCherry (a monomeric RFP) was evaluated in E. coli BL21 (DE3) with a regulated LacI promoter and lambda pL promoter hybrid. Finally, we analyzed and interpreted both successful and unsuccessful results, where we were able to define the opportunity areas as well as to provide information on the troubleshooting that can help future iGEM teams.
The creation and registry of sixteen new BioBricks™, each containing the genetic code for the expression of the target proteins:
Table 1. Parts added to the registry.
| Sequence | Accession Number | Abbreviation | Status |
|---|---|---|---|
 |
BBa_K4147000 | N/A | Registered |
 |
BBa_K4147001 | N/A | Registered |
 |
BBa_K4147002 | N/A | Transformed |
 |
BBa_K4147003 | N/A | Transformed |
 |
BBa_K4147004 | DrsB1 | Transformed |
 |
BBa_K4147006 | N/A | Registered |
 |
BBa_K4147007 | PcOSM | Transformed |
 |
BBa_K4147008 | DrsB1-improved | Transformed |
|
BBa_K4147009 | N/A | Registered |
 |
BBa_K4147010 | mCherry-LacI | Transformed |
 |
BBa_K4147011 | mCherry-Hybrid | Transformed |
 |
BBa_K4147012 | Up part of cassette for siRNA production of RXLR gene for P. capsici | Registered |
 |
BBa_K4147013 | Down part of cassette for siRNA production | Registered |
 |
BBa_K4147014 | Reverse of BBa_K525998 (RBS and promoter) | Registered |
 |
BBa_K4147015 | Reverse of BBa K864600 (Terminator) | Registered |
 |
BBa_K4147016 | RXLR siRNA | Registered |
The parts (DrsB1, PcOSM-LacI, DrsB1-improved, mCherry-LacI, mCherry-Hybrid promoter) were synthesized with the standard prefix and suffix flanking the regions of interest to facilitate restriction. The PcOSM, DrsB1 and DrsB1-improved parts were also synthesized by changing the LacI promoter to a lambda pL hybrid promoter (although these were not registered). Once the sequences were obtained, multiple cycles of PCR were performed with the objective of adding extra nucleotides on both ends to ensure a double digestion with the restriction enzymes EcoRI and PstI, to finally insert said sequences on the expression vectors ligating with MasterMix Ligase. Thanks to this, the complete expression constructs were obtained (Figure 1).
Figure 1. SnapGene® maps of A) BioBrick™ BBa_K4147008, B) BioBrick™ BBa_K4147004, and C) BioBrick™ BBa_K4147007 ligated into backbone pSB1C3. D) BioBrick™ BBa_K4147010. E) BioBrick™ BBa_K4147011.
The last two constructs (mCherry-LacI (D) and mCherry-Hybrid (E)) were created with the purpose of analyzing the different promoters and comparing the results with the predictions made through the mathematical model.
Finally, the transformation of said constructs into E. coli BL21 (DE3) was carried out by means of heat shock. Clones were then selected for plasmid extraction and visualization by electrophoresis. All this was possible to achieve during the past months by carrying out the experimentation process described next, however the detailed protocols can be found in the experiments section.
We amplified the sequences received from IDT® with the Taq Platinum polymerase at 50ºC and 55°C. After several PCR attempts, the PcOSM part with the hybrid promoter was obtained as expected (Figure 2). This allowed us to standardize the PCR protocol and add bases at both ends to later perform restriction digest.
Figure 2. PcOSM with hybrid promoter amplification. SnapGene© PCR simulation in a 1% agarose gel (1). Electrophoresis of an 0.8% agarose gel for the PCR amplification (2). A) Molecular Weight Marker Quick-Load® 1 kb Plus DNA Ladder. B) Amplification of the PcOSM with the hybrid promoter at 50°C for alignment. C) Amplification of the PcOSM with the hybrid promoter at 55°C for alignment.
The next step after standardization of the PCR protocol was to amplify the remaining sequences received from IDT. As seen in Figure 3, the sequences DrsB1-improved with both the LacI promoter and the hybrid promoter were amplified correctly at 50°C and 55°C. The PcOSM insert with a hybrid promoter was also amplified successfully at 60°C and 65°C, which meant we could proceed to ligate them into a backbone (this with a previous purification with the PureLink™ Quick Gel Extraction Kit to remove unspecified products). On the other hand, when trying to amplify PcOSM with the LacI promoter, no product appeared at either temperature.
Figure 3. Comparison between amplifications of inserts with LacI promoter and hybrid promoter in a 0.8% agarose gel. A) Molecular Weight Marker Quick-Load® 1 kb Plus DNA Ladder. B) Miniprep extraction of pJUMP28-1A plasmid. C) DrsB1-improved with LacI promoter at 50°C. D) PcOSM with LacI promoter at 50°C. E) DrsB1-improved with hybrid promoter at 50°C. F) DrsB1-improved with LacI promoter at 55°C. G) PcOSM with LacI promoter at 55°C. H) DrsB1-improved with hybrid promoter at 55°C. I) PcOSM with a hybrid promoter at 60°C. J) PcOSM construct with a hybrid promoter at 65°C.
Simultaneously we tried to purify pJUMP28-1A for the further ligation of the parts, so we did a Miniprep extraction after transformation, but the expected band wasn’t obtained on the electrophoresis gel (Figure 3 well B). After many unsuccessful attempts to recover said plasmid, we ceased to keep trying at this idea.
We amplified the DrsB1 insert with the LacI promoter again at 50°C and 55°C and the expected band size of 540 bp was obtained (Figure 4). This confirmed that we had one of the constructs ready to purify with the PureLink™ Quick Gel Extraction Kit to remove unspecific bands for subsequent digestion and ligation with the expression vector.
Figure 4. DrsB1 with LacI promoter amplification. PCR simulation on SnapGene© of PCR results in a 1% agarose gel (1) and electrophoresis of an 0.8% agarose gel for the PCR amplification(2). A) Molecular Weight Marker Quick-Load® 1 kb Plus DNA Ladder. B) PCR product of DrsB1 insert with the LacI promoter at 50°C for alignment. C) PCR product of DrsB1 insert with the LacI promoter at 55°C for alignment.
At this point we had amplified the following sequences:
PcOSM with a LacI promoter.
PcOSM with a hybrid promoter.
DrsB1-improved with a hybrid promoter.
DrsB1-improved with a LacI promoter.
DrsB1 with a LacI promoter (existing Part).
We decided to order the synthesis again with adapter sequences which would add base pairs at the ends allowing for direct digestion without amplification. Other than saving time, amplification errors would be avoided since the used polymerase wasn’t of high fidelity.
Subsequently, digestion was carried out with EcoRI and PstI restriction enzymes. Initially, we performed the ligation in the pUC19 cloning vector in order to standardize the ligation protocol. The protocol was applied when ligating DrsB1-improved, DrsB1, and PcOSM into dephosphorylated pSB1C3 and later transforming into E. coli BL21(DE3).
Additionally, the inserts of mCherry-LacI, mCherry-Hybrid, (CBD)2-DrsB1, and PcOSM without DsbA with vector pTWIST Kan High Copy were also transformed into E. coli BL21(D3).
A colony PCR was made for the transformed constructs with the GoTaq Flexi DNA polymerase to confirm the presence of the insert, and the expected band sizes were obtained. The results were visualized in a 0.8% agarose gel as shown in Figure 5.
Figure 5. Colony PCR from E. coli BL21 (DE3) transformants. Simulation on SnapGene© of PCR results in a 1% agarose gel (1) and agarose 0.8% gel electrophoresis of the complete constructs (2). A) Molecular Weight Marker Quick-Load® 1 kb Plus DNA Ladder. B) mCherry-LacI C) (CBD)2-DrsB1. D)PcOSM without DsbA E) mCherry-Hybrid promoter F) PcOSM.
We performed a Miniprep extraction to prove the presence of the DrsB1 in pSB1C3 plasmid with mCherry-LacI as the positive control.
Figure 6. Miniprep extraction DrsB1 in pSB1C3. Simulation on SnapGene© of the constructs on a 1% agarose gel (1) and agarose gel electrophoresis of the constructs(2). A) Molecular Weight Marker Quick-Load® 1 kb Plus DNA Ladder. B) mCherry-LacI uncut 3286 bp. C) DrsB1 uncut 2553 bp.
After having successful ligation we confirmed that the constructs were correctly designed and had the potential to express. In order to confirm that, we advanced on to the third experimentation round.
Isopropyl β-D-1 thiogalactopyranoside (IPTG) was used to induce protein expression. This reagent activates the Lac promoter by preventing the Lac repressor from performing its repressor activity, since it cannot bind to the operator region. Extraction with B-PER ™ was then performed to extract intracellular proteins. This strategy resulted in problems expressing the antimicrobial peptides (DrsB1-improved, DrsB1 and PcOSM), therefore, we decided to start performing the mathematical model induction assays with mCherry-LacI and mCherry-Hybrid promoter.
The induction of both mCherry inserts (with a LacI promoter and with the lambda pL hybrid promoter) was done with constructs from Twist Bioscience, both expressing a 26.7 kDa protein. These were transformed into E. coli BL21 (DE3) by means of heat shock. As shown in Figure 7, an acceptable transformation efficiency was achieved. Growth in the culture medium alone would indicate a successful transformation; however, the color of the clones is another indicator that the plasmid insertion was successful.
Figure 7. E. coli BL21 (DE3) transformed with mCherry-LacI (1) and mCherry-Hybrid promoter(2).
We later performed induction using IPTG, growing transformed E. coli BL21 (DE3) for 4 hours and adding 0 mM, 0.25 mM, 0.50 mM, 0.75 mM and 1.0mM IPTG for another 4 hours. The culture was then diluted 3:1 with LB broth added with kanamycin in order to be loaded onto a 96-well plate and read. These samples were also analyzed with an SDS-PAGE.
For the induction of the mathematical model assays, an SDS-PAGE was performed using a different construct of mCherry of known molecular weight as a control. The expected protein had a weight of 31.1 kDa due to its 2 His-tags. The induction of mCherry-LacI and mCherry-Hybrid promoter was also performed.
As shown in Figure 8, lanes D-J have pronounced bands larger than 30 kDa, whereas lanes B-C have no such bands, implying that the mCherry protein was expressed. However, there are no discernible differences between hours of induction and IPTG concentration.
Figure 8. mCherry control induction kinetic at different IPTG concentrations. Visualization through SDS-PAGE of the result. The expected band size of the mCherry control is 31.1 kD. A) Page Ruler ™ Low Range Unstained Protein Ladder. B) BL21 (DE3) without induction. C) BL21 (DE3) induced 1 mM. D) mCherry control with 0 mM 1 hour. E) mCherry control 0.1 mM 1 hour F) mCherry control 0.5 mM 1 hour. G) mCherry control 1 mM 1 hour. H) mCherry control 0 mM 2 hours. I) mCherry control 0.1 mM 2 hours. J)mCherry control 0.5 mM 2 hours.
The gel in Figure 9 shows the induction of the 31.1 kDa mCherry protein (lanes B-E) and the 26.7 kDa mCherry-Hybrid promoter protein (lanes F-J), with thick and defined bands in each case. Similarly, expression can be detected but no differences in concentrations or induction times.
Figure 9. mCherry constructs (mCherry control and mCherry-Hybrid promoter) whole induction kinetic at different IPTG concentrations. Visualization through SDS-PAGE of the results of the mCherry control and mCherry-Hybrid promoter construct induction. The expected band size of the mCherry control is 31.1 kDa and 26.7kDa for mCherry-Hybrid promoter. A) Page Ruler ™ Low Range Unstained Protein Ladder. B) mCherry control 0 mM 8 hours. C) mCherry control 0.1 mM 8 hours. D) mCherry control 0.5 mM 8 hours. E) mCherry control 1 mM 8 hours. F) mCherry-Hybrid promoter 0 mM 1 hour. G) mCherry-Hybrid promoter 0.1 mM 1 hour. H) mCherry-Hybrid promoter 0.5 mM 1 hour. I) mCherry-Hybrid promoter 1 mM 1 hour. J) mCherry-Hybrid promoter 0 mM 2 hours.
Furthermore another induction attempt (Figure 10) showed mCherry-LacI protein bands (Lane D-G) even in those samples not induced (D, F, H) while these were not found in the controls (B-C) nor in those wells loaded with the PcOSM sample (H-I).
Figure 10. mCherry-Hybrid and PcOSM without DsbA induction. Visualization through a polyacrylamide gel 12% (SDS-PAGE) of the results. The expected band size of the mCherry-Hybrid promoter and also mCherry-LacI are 26.7kDa, and 25.6 kDa for PcOSM without DsbA. A) Page Ruler ™ Low Range Unstained Protein Ladder. B) E. coli BL21(DE3) not induced. C) E. coli BL21(DE3) induced with 1mM. D) mCherry-Hybrid promoter not induced. E) mCherry-Hybrid promoter induced with 1mM. F) mCherry-LacI not induced. G) mCherry-LacI induced 1mM. H) PcOSM without DsbA not induced. I) PcOSM without DsbA induced 1mM
These results showed that there was protein expression even when no IPTG was added based on the similarities among the banding patterns at consecutive hours and concentrations, as well as its similarity with the control (E. coli BL21 (DE3)). Additionally, the strong pink color on figure 7 demonstrates protein production even after the induction protocols began.
The gels show unsuccessful obtainment of the desired protein. Since the results did not come out as expected we opted for looking for possible explanations that could help us understand the results and solutions.
After several induction attempts, we still got no results, so we decided to investigate further on the possible sources of error in the experiments. Firstly we analyzed the E. coli strain used. The BL21 (DE3) system contains the gene that codes for the T7 RNA polymerase under the control of an IPTG inducible promoter (Hernández et al., 2021). However the T7 polymerase is highly selective when heavily induced, producing up to 50% of the expected recombinant protein. Additionally, this strain is deficient in the Ion and ompT proteases, both in charge of degrading proteins during the purification process (Quiroga, 2012).
Besides the selected strain, the induction parameters were also reviewed, including the IPTG concentrations, where the kinetics of induction against time reflected the changes in concentration and the quality of the IPTG. Correspondingly, kinetics of up to 8 hours were set up, even though bibliography states that 4 hours is enough to consider a kinetic assay done. Likewise, 3 inductor concentrations (0.1 mM, 0.5 mM and 1.0 mM) were used on the constructs, using other concentrations to the assay for the mCherry constructs (0.25 mM, 0.50 mM, 0.75 mM, and 1.0 mM). On every induction experiment fresh IPTG would be prepared to achieve max quality.
Due to time constraints in the lab, we were not able to test all of our hypotheses. You can read more about this on the Engineering page.
The creation and registry of sixteen new BioBricks™.
The assembly of six new composite BioBricks: PcOSM [BBa_K4147000], PelB-(CBD)2-DrsB1 [BBa_K4147001], Expression construct for (CBD)2-DrsB1 [BBa_K4147002], PcOSM Construct with LacI regulated promoter [BBa_K4147003], mCherry construct with LacI regulated promoter for bacterial expression [BBa_K4147010], and mCherry construct with lacI regulated, lambda pL hybrid promoter for bacterial expression [BBa_K4147011]. All of them are expression systems for antimicrobial peptides.
Characterization of LacI [BBa_R0010] and Lambda [BBa_R0011] promoters.
After several experiments with the IDT synthetic sequences, we realized that the DNA was not in optimal conditions, thus soliciting a reposition of the product, and simultaneously ordering new sequences from Twist Bioscience to avoid time delays.
At the time of amplifying the constructs with PCR we had problems given that we couldn't identify the correct alignment temperatures for the primers [BBa_G1004] and [BBa_G1005]. Therefore, we did a temperature gradient of 50-65°C, identifying an optimal temperature of 55ºC where minimum unspecific products were obtained.
For weeks, we had problems with the ligation protocol. Molar ratios of 3:1, 5:1, and 7:1 were tried with no success. Following that, mass ratios of 3:1, 5:1, and 7:1 were tested. We also tried different ligases such as T4 ligase from Invitrogen and Anza™ T4 DNA ligase Master Mix with different quantities of enzymes ranging from 1 to 5 µL at [5 units/µL]. Nevertheless, we realized that the best combination of parameters consisted of a 3:1 mass proportion, dephosphorylated vector and using the Anza™ T4 DNA ligase Master Mix.
From the beginning we knew we would have setbacks, especially due to the nature of our ambitious goals and what we wanted to reach. Likewise, we are aware of the areas for improvement that we had during these eight months, as well as the future refinements that should be done experimentally in order to achieve the proposed solution. Finally, even though we did not obtain satisfactory results, these will be very useful for future iGEM teams, as well as helping us to continue analyzing the viability and scalability of our solution beyond the competition.
González, A., & Fillat, M. F. (2018). Aspectos metodológicos de la expresión de proteínas recombinantes en Escherichia coli. Revista de educación Bioquímica, 37(1), 14-27.
Hernández-Alcántara, G., García-Torres, I., & Alba-Martínez, Z. (2021). Expresión de proteínas recombinantes en un sistema heterólogo
Kesik-Brodacka, M., Romanik, A., Mikiewicz-Sygula, D., Plucienniczak, G., & Plucienniczak, A. (2012). A novel system for stable, high-level expression from the T7 promoter. Microbial cell factories, 11, 109. https://doi.org/10.1186/1475-2859-11-109
Quiroga Campano, A. L. (2012). Optimización del cultivo de Escherichia coli para la producción de cutinasas recombinantes.
