- Cloning & SDS-PAGE
For the ultimate goal of realizing the system in farmlands, we first attempted to cost down by producing peptides from E. coli to prepare for the mass production.
However, there were few people to produce antifungal peptides by E. coli because some types of antifungal peptides were long been considered to harm E. coli.
As a result, apart from predicted peptides, we also added five peptides from our search system in Parabase Website to do experiments as controls. Two of them, MiAMP1, and LA-LTP had already been produced by E. coli before, but we still tried to express them by our own system in order to know whether the system we used in the lab is able to make antifungal peptides or not.
To ensure the target gene was contained in E. coli BL21 Rosetta-gami strain, we ran electrophoresis of Taq PCR product to check the size of the inserted gene.
To ensure the target genes were contained in E. coli BL21 Rosetta-gami strain, we ran the electrophoresis of the Taq PCR products to check the sizes of the insert genes. All the biobricks were composed of the conservative promoter, RBS, and target gene. The Table1 below shows the theoretical sizes of seven BioBricks we built, and Figure 1A to Figure 1B shows the results of each electrophoresis.
Table 1: The lengh of each inserted gene.
The results of electrophoresis of 7 Taq PCR product. (A)MiAMP1(BBa_K2262001, 624 base pairs). (B)LA-LTP (BBa_K2262003, 666 base pairs). (C)Sa-AFP1(BBa_K2262005, 546 base pairs). (D) Sa-AFP2(BBa_K2262007, 546 base pairs). (E) Rs-AFP2(BBa_K2262009,
546 base pairs). (F) B-1E(BBa_K2262011, 465 base pairs). (G) Pe-4(BBa_K2262013, 432 base pairs).
We tried to get the peptide from E. coli BL21 Rosetta-gami strain, which can make disulfide bonds and often be used to express proteins.
We tried to get peptides from E. coli BL21 Rosetta-gami strain, which can make disulfide bonds and often be used to express proteins. We did SDS-page to check whether the peptides were produced. Table 2 shows the mass of each peptide. Two of them, B-1E and Pe-4, were too small to run SDS-PAGE, and the other two had already been expressed before. So, the Figure 2A to 2C just only shows the result of three peptides, Sa-AFP1, Sa-AFP2, and Rs-AFP2, that can use SDS-PAGE to confirm.
Table 2: The mass of each anti-fungal peptides and status.
Figure 2: The analysis of the antifungal peptides. (A) Sa-AFP1 No.1 to No.6. (5.7 kDa). (B) Sa-AFP2 No.1 to No.6. (5.6 kDa). (C) Rs-AFP2 No.1 to No.6. (5.6 kDa).
7 Parts have already submitted to iGEM.
For the proteins expressed, we will do the three fungal experiments to do a double check, then attempt to apply them into farmlands by the use of IoT and the disease occurrence model.
For the proteins that aren’t suitable to be expressed by SDS-PAGE, we will try other expression methods.