Introduction
For the Synfronteras team, participating in the iGEM competition represents a unique opportunity to learn and collaborate with synthetic biology, establishing laboratory conditions that allow us to develop projects on wetlab and making synthetic biology a traditional line of research at our university. Nonetheless, the pandemic of COVID-19 caused a sanitary and economic crisis in the whole world. In these exceptional conditions and aiming to achieve our goals, it was essential to think about the safety of the team members who would attend the laboratory.
In Brazil, the vaccination against SARs-CoV-2 started later than countries from Europe and North America. Therefore, our team only carried out specific activities until the availability of vaccines. When the vaccine became available, the team members that were fully vaccinated performed most laboratory activities.
Team goals
To develop the project on wetlab, the team counted on the collaboration of associate professors and researchers from our university and others around the country. These collaborations were crucial for obtaining plasmids and microorganisms that would be essential in our project and valuable to standardizing protocols and procedures. The initial activities were focused on the: preservation of microorganisms, obtaining purified plasmids and chemo-competent bacteria, transformation efficiency tests and other basic molecular biology procedures.
Project goals
Due to our limitations and the unusual conditions caused by the pandemic, the main goal of the team was to demonstrate the parts of our design that were considered essential to the project. In this sense, we chose to test our elimination module, whose main feature is using of a trypsin-activated effector molecule, antimicrobial peptide (AMP) DRS-N of the dermaseptin class, that might induce toxicity in Leishmania spp. [1]. For this, it was necessary to obtain proAMP, test its activation through trypsin-induced proteolytic cleavage and evaluate the leishmanicidal activity of AMP
Work cell
Initially, to test our elimination module, it was necessary to produce our effector molecule. Although Bacillus subtilis is more suitable for AMP production, due to the unavailability of an electroporator and the difficulty of transforming B. subtilis by other transformation methods (eg. heat shock), the bacterium Escherichia coli was chosen as the host to obtain proAMP [2]. Furthermore, E. coli also presents advantages related to ease of cultivation and chemo competency induction, crucial factors in view of the limited time available for laboratory experiments.
Assembly
Knowing that we would use Gibson assembly to assemble our parts, we designed our gBloks to be as modular as possible, first we selected primers suitable for use in our expression plasmid pBBR1MCS-2 and cloning plasmid pUC19, to produce common ends. We used benchling platform to generate overlap regions, which was the same for both plasmids, so we used the same overlap in the design of all our inserts. Additionally, we standardized to put biobrick prefix and suffix sequences before each overlap to enable biobrick assembly.
Figure 1: The primers designed can be used to linearize both our expression and cloning plasmids, and as it's in the lacZ region it is possible to be used in other plasmids.
Figure 2: The overlaps designed were used in all insert fragments and, this way, can all be assembled in both linearized plasmids or others who used the previously designed primers.
All parts designed maintained a modular character and were divided into two modules, the expression/export module and the coding module, allowing its use in different contexts except for the main (G1) and reporter (G0) circuits, designed for a specific objective.
Figure 3: Our module of expression/export and the proAMP module can be freely combined among themselves.
After obtaining the linear pBBR1MCS-2, we used the NEBuilder HiFi DNA Assembly Cloning Kit to assemble the G1 and G0 parts and cloning in NEB 5-alpha Competent E. coli (C2987H), and reclone at E. coli BL21 (D3). The protocols used for this experiment, P006A, P006B, P008A, P009A, P010A, P011A, P002A, can be found on our protocol page.
To confirm the assembly, we chose used PCR to amplify the inserts and the electrophoresis to visualize it, using as template plasmid miniprep and colonies from selection plates.
ProAMP obtainment
To produce our proAMP, we used the expression strain Escherichia coli BL21 (D3), which is inducible by IPTG, and the expression vector pBBR1MCS-2, with our main circuit (G1) T7-LacO Promoter + RiboJ + RBS_0034 + 6xHis-tag + proDRS-N1 + double Terminator (BBa_K4075008).
After the production, we carry out the obtention of the effector molecule by collecting the extracellular portion of the cultivation and the periplasmic extract of cells. The protocols used for this experiment, P012A, P013A, can be found on our protocol page.
Trypsin proteolytic cleavage
According to Guilherme Brand, responsible for the characterization of DRS-N1, the propeptide is a trypsin-like target, which is evident when we observe the cleavage site between the acidic piece and the AMP. Nonetheless, there are other spots in the AMP that are susceptible to trypsin action, as shown:
Figure 4: The cleavage sites were predicted by the peptide cutter tool from ExPASy. Both KR amino acids are in the propeptide section and are more likely to be cleaved by trypsin; furthermore, there are other cleavage sites, which indicates the possibility of partial digestion.
We believe that for this characteristic, the activation of the proAMP may occur by partial digestion, so the amount of trypsin will be determinant for the AMP activity. We treated the extracellular and periplasmic obtention products with three different concentrations of trypsin 0.5, 1.0 and 1.5 mg/mL to test this aspect. According to previous studies using trypsin digestion, these concentrations were chosen[3][4]. After that, the final step of our experiments allows us to evaluate which conditions are ideal for the AMP activation.
Partial digestion can be an advantage knowing that the trypsin inside the sand-fly midgut is not fully available due to its role in digesting the blood meal. Protocol used: P014A
Leishmanicidal assay
To the final step of our experiments, we verified the leishmanicidal activity of our AMP and how the trypsin treatment worked on activating it. For this purpose, cultures of Leishmania infantum were incubated with the products derived from the bacterial culture portion and the periplasmic extract of cells, both previously treated with trypsin. The Leishmania sp. were observed after five days of incubation. Protocol used: P015A.
Future Perspectives
For 2021 participation on iGEM, the only aspect tested in wetlab were characteristics from the elimination module. However, there are several questions still to be solved and demonstrated. Many studies are still needed once this project uses a complex implementation strategy in a new application.
Besides testing the elimination module, it would be needed to verify the remaining modules on wetlab, to demonstrate engineering success and complete the engineering design cycle (Design + Build + Test + Learn + Design) on these modules too.(To see the modules check our design page). For the detection module, the major issue was the characterization of endogenous miRNAs to test the feasibility of the designed toehold switch. Its function could be demonstrated by synthesizing the miRNAs candidates in a second circuit evaluating the activation of the switch.
On the Protection device module, it would be necessary to induce the chassis to its sporulated form and test it in the presence of L-alanine, which would turn it for its vegetative form, making it available for detection and expression of other circuits.
The Killswitch module requires an optogenetic tool to expose the chassis to light, as, after transitioning to its vegetative form, our killswitch system becomes sensitive to expression triggers. This way, we could obtain quantitative data on the light exposure needed to kill the bacteria.
Beyond testing the modules, it would be necessary to validate the chassis, using a closed system to deliver it transformed with a reporter gene to sand-fly in sugar baits. In the next step, a sample of the population tested would be evaluated to see if Bacillus subtilis could colonize sandfly midgut.
Our team believes that answering these questions could help enlighten the pathways of paratransgenesis through synthetic biology and, hopefully, this work will be continued, synthesizing a new future.
References
Brand, G. et al. (2013). The Skin Secretion of the Amphibian Phyllomedusa nordestina: A Source of Antimicrobial and Antiprotozoal Peptides. Molecules 2013, Vol. 18, Pages 7058-7070, 18(6), 7058-7070. https://doi.org/10.3390/MOLECULES18067058
Ji, S. et al. (2017). Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700. Scientific Reports 2017 7:1, 7(1), 1-10. https://doi.org/10.1038/srep40587
KAWASAKI et al. (1986) et al. Limited Digestion of Cahnodulin with Trypsin in the Presence or Absence of Various Metal Ions. The Journal of Biochemistry, v. 99, n. 5, p. 1409-1416. https://academic.oup.com/jb/article-abstract/99/5/1409/1011673
VISHWESHWARAIAH et al. (2021)Rational design of hyperstable antibacterial peptides for food preservation. npj Science of Food, v. 5, n. 1, p. 26. https://www.nature.com/articles/s41538-021-00109-z