Introduction
Our team understands that demonstrating the project's applicability as a whole would be ideal for presenting a suitable proof of concept. However, due to our limitations and the unusual conditions caused by the pandemic, our goal was to demonstrate the parts of our design that we considered critical for the project. The elimination module is responsible for Leishmania spp. inactivation, which is essential for the Biopank functionality. Therefore we attempt to demonstrate it. The description of each step is available on our experiments page.
Assembly
At first, we struggled a little to obtain our expression plasmid pBBR1MCS-2 linearized. Before getting the Monarch Miniprep Kit (New England Biolabs, USA), we carried out an alkaline lysis miniprep, however did not have RNase to clean up the RNAs. After using the miniprep kit, a cleaner result was obtained, but as the plasmid is a low copy one, our yields were not high.
After the obtention of the purified plasmid, PCR reactions for linearization were started. Still, due to a low yield of DNA fragments on the gel and the lack of a proper DNA ladder, we could not be sure of having a positive result, leading us to digest lambda DNA and use it as a ladder. All the steps were repeated to get the purified linear plasmid. Later, we realized that the lower yield could be related to the melting temperature used, since there was a difference between the Tm calculator temperature and the one provided by NEB Tm calculator.
Figure 1: Lanes three to seven results from different PCR reactions to linearize the pBBR1MCS-2 plasmid. The first lane with the EcoRI digested lambda DNA worked as a ladder since the available one was impracticable. Using literature information about lambda DNA, we could estimate the molecular weight of fragments, therefore concluding the successful linearization of the plasmid for its size of 5148 bp.
We successfully assembled the two constructs selected in our experiments: the G1 containing the CDS of the proAMP and G0, with the reporter gene of mCherry. The assembly was confirmed in E. coli DH5-alpha by PCR with miniprep and colonies from selection plates amplifying the inserts followed by electrophoresis.
Figure 2: The first lane with the product PCR from the G0 synthesis (gBlock) amplification is a positive control, leading us to find the positive samples at lanes 2-3 and 9-10, from E. coli DH5-alpha, assembled with G1. Those samples were obtained by PCR amplification of miniprep and colony PCR. The difference between the positive control and positive samples is justified because G0 is 1179 bp long and G1, 614 bp.
The miniprep from E. coli DH5-alpha was used to clone E. coli Bl21(D3). The cloning confirmation was done by colony PCR amplifying the inserts followed by electrophoresis. Additionally, other samples from E. coli DH5-alpha assembled with G0 were picked to be confirmed. All the assemblies were cultivated and preserved.
Figure 3: This new conformation electrophoresis used the same positive control. The G0 assembly on E. coli DH5-alpha was confirmed as positive on the third lane. At lanes four to six, colony PCR from E. coli BL21 transformed with G1 assembly was confirmed as positives. A linear sample of pBBR1MCS-2 was used as a negative control.
proAMP production
The expression of our proAMP is inducible by IPTG, and it has the peptide signal OmpA [1]. A specific timing of induction and cultivation conditions can favor the exportation of the proAMP to the periplasmic and extracellular region, this would save some time and facilitate the purification process[2][3]. We planned to identify the production of the AMP by immunoblot using the 6xHis-tag as target, but unfortunately we got our parts later than expected and couldn't perform it.
In this shorter time to present results, we decided to obtain our effector molecule from extracellular and periplasmic media for an indirect demonstration through he inactivation of Leishmania infantum.
AMP activation and leishmanicidal activity
The leishmanicidal assay was incubated for five days, under the six samples of activated AMP from the last step and control samples, including proAMP without enzymatic treatment. We expected to count Leishmania infantum viable cells to determine their growth during the incubation time. Unfortunately, the medium used wasn't in good condition to properly cultivate the cells, and contamination was found. Due to these factors, the experiment was interrupted.
It took longer than expected to get the gene synthesis and assemble the parts, so there was no more time to restart the experiment and get results before the wiki freeze. We hope to present these results later in 2021 or at upcoming participation.
References
Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, 5:1200, 2016. Article.
Peng, L., Xu, Z., Fang, X., Wang, F., & Cen, P. (2004). High-level expression of soluble human β-defensin-2 in Escherichia coli. Process Biochemistry, 39(12), 2199-2205. doi:10.1016/j.procbio.2003.11.011. Article.
Rollan, C H. et al. Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli. Technical University of Denmark, 2020. dx.doi.org/10.17504/protocols.io.bdr2i58e. Protocol.