Part:BBa_K4593022

Integrative device of endolysin production-release module

Usage and Biology

This composite part is a device that allows the E. coli to express S. aureus targeting endolysin LysDZ25 when induced by xylose, and express self-lysing complex Spn1s_LysRZ to release the endolysin when induced by IPTG. By first inducing E. coli containing this part with xylose and then with IPTG, the collected lysate will contain LysDZ25 and can be used to eliminate S. aureus.

For detailed characterizations of its subparts, see the links below:

LysDZ25: BBa_K4593000

Spn1s_LysRZ: BBa_K4593001

BNDS-China-2023

Our team constructed this part to test the feasibility of integrating the endolysin expression circuit with the endolysin release circuit. The two previous engineering cycles had successfully demonstrated that endolysin LysDZ25 is capable of lysing Staphylococcus aureus and the self-lysing complex SPN1S_LysRZ is capable of lysing the engineered E.coli itself. Thus, this part could achieve the production and release of endolysin in one plasmid. The genetic circuit of this part is shown below.

Figure 1. Abbreviated gene circuit of DZ25_Spn1S_LysRz-PET28a. (RBS and terminators are omitted, created by Biorender)

Characterization of lytic activity to S. aureus when expressed in E. coli

methods and result

A Lysis ability assay was performed to investigate the lysis ability of E. coli with the plasmid LysDZ25_SPN1S_LysRz-PET28a to S. aureus. After being inoculated to fresh LB medium and the culture’s OD600 reaches 0.6, E. coli BL21(ED3) with the plasmid LysDZ25_SPN1S_LysRz-PET28a was cultured in LB K+ culture under 0.5% (w/v) xylose for 4 hr and then induced by 0.5mM IPTG for another 4 hr. The resulting culture is centrifuged at 10000xg and the supernatant is collected. S. aureus was cultured in TSB medium for 8 hr, 1 ml of the resulting TSB culture of SA was transferred into a 1.5 ml centrifuge tube and centrifuged at 3000 rpm for 10 min, and 900 μL of the supernatant was discarded and the S. aureus pellet was resuspended with the rest of the supernatant. The resulting concentrated TSB culture was spread gently across an S. aureus chromogenic media plate. Then two 5 mm * 5 mm square holes in the agar were created along the plate's diameter at approximately 1/3 diameter and 2/3 diameter. 20 μL of the E. coli supernatant collected as mentioned above and 20 μL of LB K+ media without bacteria culture were added to the two holes on the S. aureus chromogenic media plate coated with S. aureus right after the holes were created. The plate was then cultured overnight; the observations are shown in Figure X.1.

Figure 2. S. aureus chromogenic media plate coated with S. aureus. 20 μL of LB K+ culture of E. coli with the plasmid LysDZ25_SPN1S_LysRz-PET28a and induced by xylose and IPTG was transferred to the left hole; 20 μL of clear LB K+ media were transferred to the right hole

Observations show an obvious inhibition zone surrounding the left hole, while the surroundings of the right hole do not show observable differences compared to the rest of the plate. This indicates that the engineered E. coli with the plasmid LysDZ25_SPN1S_LysRz-PET28a is capable of lysing S. aureus after our proposed endolysin expression-self lysing procedure.

Conclusion

After the test, we preliminarily concluded that the integration of endolysin production module and self-lysing release module in one plasmid LysDZ25_SPN1S_LysRz-PET28a are successful and the transformed post-induction E. coli culture supernatant are capable of lysing bacteria S. aureus. However, the conclusion that the lysis effect results from LysDZ25 endolysin production can not be given since the control used is clear LB K+ media making us unable to rule out the possibility that other contents originate from E. coli cell lysate have bactericidal ability against S. aureus. Also, the conclusion that SPN1S endolysin and Rz-like spanin solely contribute to the lysis of E. coli and release of contents may not be accurate since the centrifugal process with 10000xg might lysis some engineered E. coli and spread its contents into the supernatant, causing a chain reaction. But overall, this engineering cycle provides verification that the engineered E. coli with the production and release of endolysin integrated could possibly lysis the bacteria S. aureus, which provides direction for further plans of engineering E. coli that can sense and lysis S. aureus automatically.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2672
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 3567
  Illegal XhoI site found at 4510
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 5009
  Illegal AgeI site found at 5147
  
• 1000
  COMPATIBLE WITH RFC[1000]