Team:HKUST-Hong Kong/Assembly

To assemble the three modules together and transform our construct into B. subtilis, an integration plasmid, pDG1661, from BGSC was employed. There are several reasons why we decided to use this vector.

1. Plasmid instability in B. subtilis:
Plasmid instability is a phenomenon observed in DNA recombination with Gram-positive hosts. Frequently occurring structure alterations and loss of plasmids prompted the study in integration vectors. In our project, we utilize an integration plasmid. The whole construct is inserted into its multiple cloning site and integrated into the B. subtilis genome through homologous recombination. This integration can help stabilize the recombinant DNA and generate relatively genetically stable bacteria.

2.Biosafety:
Given the possibility of horizontal gene transfer between bacteria within normal flora in the gut, integration plasmid is used in order to reduce the chance of plasmid loss and the transfer of antibiotic resistant genes to other microflora in gut.

The integration vector we used in our project, pDG1661, facilitates the integration through the 5’ and 3’ segment of B. subtilis amyE gene flanking at the two ends of the multiple cloning site. A cat gene encoding chloramphenicol acetyl tranferase is located within the integration area for B. subtilis transformation selection. Containing only a replication origin for E. coli, this vector needs to be replicated in E. coli first and selected through ampicillin resistance obtaining from the bla gene on this vector. After replication in E. coli, the recombinant DNA plasmid is transformed into B. subtilis and only the ones that have undergone plasmid integration can survived under chloramphenicol selection, forming colonies. The integration can be further verified through the starch test. As shown in the photo below, since the cells without pDG1661 integrated can utilize starch, they are surrounded by a white halo after overnight incubation on a LB plate that has been supplemented with starch. This can be visualized by adding gram iodine after overnight incubation. However, since the integration will split the amyE gene, the defective B. subtilis can no longer use starch, and no white halo can be observed after adding iodine.

Photo: Starch test for plasmid integration

3. Final Assembly:

The final recombinant DNA plasmid we get is shown in Figure above. Driven by Pveg promoter, RPMrel peptides can be highly expressed and displayed on the cell wall after fusing with the lytC displaying system. Two protein coding genes, Bmp2 and toxin ydcE are under the control of a xylose inducible promoter PxylA so that after binding, the induction of xylose can trigger the expression of both toxin and anti-tumor chemokine at the same time. Ptms, a weak constitutive promoter coupled with the antitoxin ydcD is then inserted into the same vector as well in order to limit BMP2 production through the need to maintain comparable toxin-antitoxin production rates.

Since pDG1661 contains one XbaI cutting site and three PstI cutting sites outside its multiple cloning site, inserts cannot be ligated via standard bio-brick assembly methods. Instead, we utilize vector pBluescript II KS(+) to add a BamHI cutting site after PstI and inserting all our construct through EcoRI and BamHI site.