Team:Montpellier/WetLab Peptides

Design

General design

Each of our constructions contained RpsU promoter [1] which is a Lactobacillus jensenii strong promoter. This RpsU sequence also contains the putative sequence for the RBS. The same scaffold is used for all of our designs to facilitate their constructions [2]. We added spacers to all of our constructions to unable easier use of the sequence and separation of the different genes of the sequences. The spacers are of 40bp to facilitate cloning by Gibson assembly. We used two Terminators to our sequences :BBa_B0014 & BBa_B0015 to ensure the stopping of the transcription. Our constructions were assembled in the pLEM415 vector by Gibson Assembly method. pLEM415 is a plasmid that works in Lactobacilli species but it’s not specific to L. jensenii [3].

For the next design schemes the general legend is presented on figure 1:

Figure 1: Explanations for the design schemes

LL-37

For the LL-37 sequence we used the RpsU promoter and the sequence coding the LL-37 peptide.

Figure 2: Design of the sequence coding the LL-37 protein with the RpsU promoter.

Figure 3: Design of the sequence coding the LL-37 protein with the pHyperSpank promoter.

SubtilosinA

Figure 4: Design of the sequence coding the Subtilosin protein with the RpsU promoter.

Figure 5: Design of the sequence coding the Iron Sulfur Cluster with the RpsU promoter.

Lacticin 3147

This circuit was made from 2 native genes of Lactococcus lactis ltA1 and ltnA that express Lacticin peptide. Also, the design contains Lacticin-post-transcriptional regulator ltM1 and M2. A promoter orthogonal was used : ptsH and differents spacer taken from igem_parts.

Figure 6: Design of the sequence coding the Lacticin 3147

Nisin

The nisin pathway is complex and really difficult to design in E.coli or Gram positive. Indeed, we need to encode 12 genes to produce and secrete nisin. Kong & Al sent us the plasmid pWK6 (20 kB) to insert into E. coli [4]

Experiments

We had issues for synthesizing the different sequences, due to their complexity and length the two companies that we contacted were not able to synthetize them even in multiple fragments. Consequently, Lacticin and Subtilosin were not used for the rest of the project. Only the sequence for LL-37 was used.

The part coding LL-37 peptide was successfully cloned in E. coli.

We checked the insertion of the vector containing this part by PCR and electrophoresis (figure 7).

Figure 7: Gel after electrophoresis of colony PCR of pLEM415 plasmid with different inserts. For each insert two colonies were picked.

For LL-37 the second colony (15) contained the insert. The sequencing proved that the sequence was correct.

This peptide was too small to do SDS-Page to detect its production. We decided to use our sperm motility assay as a way to detect its activity. The experimental designs can be found in the analysis of sperm motility section. Unfortunately, we were not able to do the experiments for LL-37 activity assay after finishing to test and validate our experimental protocol.

Summary

	Designed	cloned	Characterized
LL37	YES	YES	NO
Subtilosin	YES	NO	NO
Lacticin	YES	NO	NO
Nisin	NO	YES	NO

Figure 8: Experiments that we carried on

References
[1]	Liu, X., Lagenaur, L. A., Simpson, D. A., Essenmacher, K. P., Frazier-Parker, C. L., Liu, Y., ... & Lee, P. P. (2006). Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrobial agents and chemotherapy, 50(10), 3250-3259.
[2]	Guiziou, et al., (2016). "A part toolbox to tune genetic expression in Bacillus subtilis" Nucleic Acids Res. 2016 Sep 6;44(15):7495-508. doi: 10.1093/nar/gkw624. Epub 2016 Jul 8..
[3]	Bao, S., Zhu, L., Zhuang, Q., Wang, L., Xu, P. X., Itoh, K., ... & Lin, J. (2013). Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats. PloS one, 8(3), e60007.
[4]	Wentao Kong, David R. Meldgin, James J. Collins and Ting Lu (2018). Designing microbial consortia with defined social interactions Nat Chem Biol821-829. doi:10.1038/s41589-018-0091-7