Latest revision as of 15:37, 12 October 2023

Sequence and Features

PsrfA-sulA

This part is composed of PsrfA(BBa_k4614202), an autoregulatory promoter derived from Bacillus subtilis 168 and sulA(BBa_K4614201),which encodes SulA，an endogenous cell division inhibitor from Escherichia coli K12. Additionally, PsrfA sequence contains RBS itself, so there is no need to insert any RBS sequence during construct. CAU-China 2023 used this part to construct a cell-density-dependent expression system in E.coli (BL21) in the filamentation module of our project, thus enabling engineered bacteria to express SulA without any inducer and start filamentation process automatically. We inserted this part into pJUMP46-2A vector and obtained E.coli BL21 harboring recombinant plasmids. The recombinant strain was cultivated for over 24 hours and examined by optical microscope after stained with crystal violet.

Fig. 1 Division arrest by quorum - sensing induction through PsrfA of SulA. The strain used was E.coli BL21 that carries sulA gene under PsrfA promoter.

As shown in Fig.1, it is suggested that sulA under PsrfA promoter is sufficient to cause filamentous growth of engineered bacteria. And for further verification of the autoregulation capacibility of PsrfA, we transferred 0.6 mL of the preculture of recombinant strain E.coli BL21 to 10 mL sterile tubes that contained 100 mL of LB liquid medium and cultivated for 24 h, during which the culture was sampled every 3 h. Samples are made by harvesting cells by centrifugation, washing pellet with ddH2O once and resuspended in an appropriate diluted ratio.Smears were photographed by optical microscope to examine the cell growth (Fig. 2).

Fig. 2 Image of cell growth of E.coli BL21 containing pJUMP46-2A-PsrfA-sulA.Cultures are priodically sampled to examine filamentous growth by optical microscopy.

It can be seen from Fig. 2 that the length of engineered bacteria,as well as the proportion of filamentous cells, had slightly increased as cultures grew expotentially. Yet，the expression characteristic of this composite part in chassis bacteria has not been quantified.

References

[1] Guan C, Ma Y, Chen X, et al. Broad-host-range application of the srfA promoter from Bacillus subtilis in Escherichia coli[J]. J Microbiol Methods, 2020,168:105798.

[2]Schoemaker, J M et al. “Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death.” Journal of bacteriology vol. 158,2 (1984): 551-61. doi:10.1128/jb.158.2.551-561.1984

[3]Mukherjee, A et al. “Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli.” Proceedings of the National Academy of Sciences of the United States of America vol. 95,6 (1998): 2885-90. doi:10.1073/pnas.95.6.2885