Results | E.Lectrode

Cloning

The proposed workflow for creating the pilus expression vectors involves several key steps. The type IV pilus assembly genes will be amplified from the E. coli K12 genome using PCR (Fig. 1). These amplified fragments, along with a synthetic gene block containing the Geobacter sulfurreducens pilus (GsPilA), will then be divided into two groups and assembled into two separate pBbE1k sub-plasmids using Gibson assembly (Fig. 2). The genes in the first sub-plasmid will include GsPilA, hofB-hofC, and hofM-hofN-hofO-hofP-hofQ, while the second sub-plasmid will contain ppdA-ppdB-ygdB-ppdC and gspO. Finally, these fragments will be amplified from the sub-plasmids and assembled into the final pBbE1c vector through Gibson assembly, resulting in a complete plasmid capable of expressing the pilus.

Figure 1. Type IV pilus assembly gene fragments from K12. The four gene fragments of the type IV pilus assembly machinery to be amplified from the K12 genome, with Gibson assembly overhangs added for subsequent cloning steps. The fragments are: (A) hofB-hofC, (B) hofM-hofN-hofO-hofP-hofQ, (C) gspO, and (D) ppdA-ppdB-ygdB-ppdC. Created with BioRender.com

Figure 2. Pili expression vectors. The gene fragments GsPilA, hofB-hofC, and hofM-hofN-hofO-hofP-hofQ will be assembled into one pBbE1k sub-plasmid (A), while the fragments ppdA-ppdB-ygdB-ppdC and gspO will be assembled into another pBbE1k sub-plasmid (B). The two resulting fragments from these sub-plasmids will then be combined and assembled into the final plasmid using Gibson assembly (C). Created with BioRender.com

The BglBrick vectors pBbE1c-RFP and pBbE1k-RFP were streaked out for single colonies. Red/pink colonies were observed, indicating successful expression of the RFP reporter gene (Fig. 3). A random colony was chosen from one of the pBbE1c-RFP plates and the pBbE1k plate to inoculate liquid cultures and placed in the 37℃ incubator overnight.

Figure 3. Streaked out cells with BglBrick vectors for single colonies for overnight cultures. Cells containing the BglBrick vectors pBbE1c-RFP (A and B) and pBbE1k-RFP (C) were streaked out onto LB plates containing either chloramphenicol or kanamycin.

Our original vector design (Fig. 2), where a single promoter controlled the expression of the entire pilus assembly system, raised some concerns. Redesigning the primers to insert a promoter in front of each operon was not feasible due to time constraints. However, because our initial plan involved subcloning, the overhangs are compatible with other BglBrick vectors. This compatibility allowed us to select a different BglBrick vector for one of the sub-plasmids, enabling co-transformation of the vectors. As a result, we could place a promoter in front of two operons (Fig. 4). This approach provided a practical solution, improving the regulation of gene expression without requiring extensive redesign.

Figure 4. Modified pilus assembly vector design. The sub-plasmid backbones were changed from pBbE1k to pBbA1k (A) and pBbE1c (B) to optimise the system, enabling direct co-transformation of the sub-plasmids without the need for prior assembly into a single plasmid. This new design incorporates two promoters for enhanced gene expression control. Created with BioRender.com

The pBbA1k vector was streaked out for single colonies and red/pink colonies were observed, indicating successful expression of the RFP reporter gene (Fig. 5). A random colony from the plate was selected to inoculate a liquid culture. Since we did not have the overnight culture of pBbA1k prepared on the day we intended to miniprep the pBbE1c vector, we quickly set up the overnight culture and proceeded with the miniprep as planned.

Figure 5. Streaked out cells with pBbA1k vectors for single colonies. Cells containing the BglBrick vector pBbA1k were streaked out onto LB plates supplemented with kanamycin.

After the miniprep, we used a NanoDrop to measure the DNA concentration of the samples (Table 1), allowing us to calculate the volume of vector needed for the restriction digest with BamHI and NdeI.

Table 1. NanoDrop results after minipreparation of vectors from overnight cultures.

Vector	Concentration (ng/μl)
pBbE1c	179.5
pBbE1k	118.9

The digested vectors and PCR products containing the amplified type IV pilus assembly system gene fragments from K12 were loaded onto a 1% agarose gel and ran at 130V for 30 min. The results were largely as expected, except that no band was observed for pBbA1a (Fig. 6). This sample, provided by an advisor, was intended to be used in place of pBbA1k if the digest had worked. However, due to the absence of a band, we proceeded with the original plan to use pBbA1k.