Our system provided a way to regulate microbiota to an ideal balance . Since we focused our model on the excess PSB in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the Bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed BioBricks that contain sequences of these peptides. All the experiments we performed with BioBricks are mentioned below.
Team:NCTU Formosa/Wet Lab
BioBrick
The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a intein-CBD (chitin binding domain) tag behind bacteriocin to better purify our peptide.
Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD
Bacteriocin
It is a peptidic toxin produced by bacteria that inhibits the growth of similar or closely related bacterial strains, but does not damage the bacteria themselve by specific immune proteins. It is heat stable and shows its antimicrobial activity in various temperatures and pH levels. Unlike most antibiotics, which are secondary metabolites, bacteriocins are ribosomally synthesized and sensitive to proteases while generally harmless to the human body and surrounding environment.
Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of gram-positive bacteria. The receptors on the target membrane can determine the specificity of bacteriocins, and the insertion of bacteriocins is driven by proton motive force driven.
Figure 2: Mechanism of bacteriocin
Purification
Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides.
Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.
Figure 2: Purification process.
Figure 2: Procedure of intein-mediated protein splicing
1. Protein expression
Cloning:
We cloned the insert gene into pSB1C3 backbone and transform into E. coli DH5α.
These have been submitted to iGEM.
Expression:
We expressed the proteins by E. coli ER2566 and E. coli BL21 Rosetta-Gami.
SDS-PAGE:
We checked the expression result through SDS-PAGE.
2. MIC test
Inhibition zone:
Minimum Inhibitory Concentration Broth Microdilution:
3. Growth curve experiment
4. Soil experiment
