Difference between revisions of "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 (ref Guiziou et al., NAR 2016). 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. Plem 415 is a plasmid that works in Lactobacilli species but it’s not specific to L. jensenii [2].

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

LL-37

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

SubtilosinA

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.

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 [3] (Figure 7).

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 8).

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

Table of the three constructs: