Part:BBa_K3740042

Chimeric Bacteriocin SE

Description

We replaced the nuclease domain of pyocin S2 with that of colicin E3 to produce a chimeric bacteriocin termed SE.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2407
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2407
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2407
  Illegal BglII site found at 2112
  Illegal BamHI site found at 1684
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2407
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2407
  Illegal NgoMIV site found at 1483
  Illegal AgeI site found at 1573
  
• 1000
  COMPATIBLE WITH RFC[1000]

2021 SZPT-China

Biology

The receptor domain, translocase domain of pyocin S2 and the nuclease domain of colicin E3 are fused together to get a new chimeric bacteriocin termed SE. The antipseudomonal SE protein can target and inhibit P. aeruginosa, even including those harbouring a S2 immunity protein IMM_S2.

Figure 1. SE domain and source of antipseudomonal drugs

Usage

The chimeric bacteriocin SE (BBa_K3740042) was constructed with an inducible expression vector to form the SE-PET28A expression vector, and transferred into E. coli BL21 to verify its function.

Characterization

1.SDS-PAGE

<p>Method: IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein expression in E. coli BL21 and subsequently incubated at 25℃ for 12 hours. Supernatant containing SE or S2 proteins was obtained by sonication and centrifugation respectively. The crude protein extract was loaded into SDS-PAGE gel and stained by Coomassie Blue to verify the presence of target proteins.

After induction by IPTG, S2 and SE protein expression in S2-PET28A-BL21 (lane 2) and SE-PET28A-BL21 (lane 3) has been identified. By contrast, target proteins were not expressed in the strain with S2-PET28A-BL21 (lane 1) and SE -PET28A-BL21 (lane 4) in the absence of IPTG, indicating that expression of S2 and SE proteins can be successfully induced in E. coli transformed with S2-PET28A and SE-PET28A, respectively.

Figure 2. SDS-PAGE analysis of protein expression in engineered E. coli BL21. Lane 1, S2-PET28A-BL21 without IPTG induction; Lane 2, S2-PET28A-BL21 with induction by 0.1mmol/mL IPTG; Lane 3, SE-PET28A-BL21 with induction by 0.1mmol/mL IPTG; Lane 4, SE-PET28A-BL21 without IPTG induction.

2. SE protein targeting verification

Method: Supernatant containing SE or S2 protein was respectively mixed with the culture of Pseudomonas aeruginosa PAO1, P. aeruginosa PAO1Δ1150-1151(PAO1 Δ1150-1151, PAO1 knocked out of S2+imm_S2) and E. coli MG1655. PBS was used as a negative control. Inhibitory effect of supernatant containing SE or S2 protein on the P. aeruginosa growth was determined by measuring OD600 using SYNERGY H1 micro-plate reader.

We found that the OD600 values of both P. aeruginosa strains cultured with SE protein, were much lower than that of the control group PBS. This proves that SE protein has antipseudomonal properties. In contrast, pyocin S2 could only act on the immune-deficiency strain PAO1∆1150-1151. This proves that SE protein has a broader antipseudomonal spectrum than S2. Meanwhile, the OD600 value of E. coli MG1655 cultured with SE protein was a little higher than that of the control group PBS. This indicates that SE protein does not work on E. coli. This result indicated that the fusion protein SE can specifically kill P. aeruginosa.

Figure 3. Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and E. coli MG1655 treated by the supernatant from S2-PET28A-BL21 or SE-PET28A-BL21 lysates for 790 minutes. PBS was used as the negative control.

References

[1] Gupta S, Bram E E, Weiss R. Genetically programmable pathogen sense and destroy. [J]. Acs Synthetic Biology, 2013, 2(12):715-723