Header Image

Index

1. yedQ 2. golS 3. mazF

Contribution Introduction

1. yedQ BBa_K4242004


YedQ is a diguanylate cyclase from Escherichia coli. It is registered in 2022 by CUG-China, which works with the phosphodiesterase (YhjH) modulated the in vivo c-di-GMP levels.

The expression of the yedQ gene increased the intracellular c-di-GMP concentration and resulted in the enhanced secretion of EPS. We design the expression vector 'pYDT-Ptac-yedQ' in E.coli BL21 and Acidithiobacillus ferrooxidans to characterize its function.

Fig 1. The gene circuit of expression vector 'pYDT-Ptac-yedQ'

We examined the role of yedQ by overexpressing yedQ and measuring biofilm formation in overexpressing engineered bacteria versus c-di-GMP content in bacteria.

Fig 2.(A) A. ferrooxidans pore plate biofilm formation column.(B) Escherichia coli pore plate biofilm formation column.(C) Schematic diagram of c-di-GMP content of engineered E.coli BL21 pYDT-0053, pYDT-1379, pYDT-1373, pYDT-yedQ. (D) Indicates the standard curve of c-di-GMP.


By overexpressing the yedQ gene, we found that A. ferrooxidans and E.coli BL21 formed thicker biofilms. yedQ also showed the highest concentration of c-di-GMP in E.coli BL21 strains, suggesting that high levels of c-di-GMP can regulate the formation of thicker biofilms.


2. golS BBa_K4468005


GolS is a gold-binding MerR family transcription factor found in Samonella sp., which enables the specific binding of Au[III] and triggers the transcription of downstream genes through the promoter Pgol. It was first registered in 2020.

Fig 3. The gene circuit of expression vector 'pYDT-golS-Pgol-gfp'


In order to quantify the specific to Au[III], we designed the expression vector 'pYDT-golS-Pgol-gfp' in E.coli BL21 to characterize its function.


Fig 4. (A) Plot of normalized fluorescence intensity of engineering bacteria E.coli BL21 in different metal ions. (B) Plot of normalized fluorescence intensity of engineered bacteria E.coli BL21 in different metal ions at 12 hours.


In the experiment, we added different metal salt solutions, HAuCl4, NiCl2, CdCl2, CuSO4, ZnCl2, to the culture system of the engineered bacterium E.coli BL21, and the ion concentrations of metal ions Au3+, Ni2+, Cd2+, Cu2+ and Zn2+ were all 20 μM.


By detecting the normalized fluorescence intensity of the engineered bacteria in different ion cultures. We found that the fluorescence intensity of 20 μM Au3+ culture was significantly higher than the fluorescence intensity produced when other metal ions were cultured. This indicates that the gene line golS-Pgol-gfp can respond specifically to Au[III].


3. mazF BBa_K1096002 and Inverter BBa_Q04510


MazF is toxin protein in MazF-MazE, toxin-antitoxin module. It was first registered in 2013 and used as an mRNA endonuclease. It kills bacteria without cracking them, which will directly influence the value of OD600.

Inverter is mainly composed of cI repressor from E.coli phage lambda and PR promoter which is inhibited by cI repressor. It can reverse the inducer action of the upstream promoter. It was registered in 2003.

In our project, we expect engineered bacteria to survive and carry out biomining activities only in environments containing gold, so we constructed bacterial suicide switches using tandem gene wiring of gold combined with specific transcription factors, Inverter and mazF.


Fig 5. The gene circuit of expression vector 'pYDT-golS-Pgol-cI-PR-mazF'


In order to quantify the function of inverter and the toxicity of mazF, We designed gene circuits to express inverter and mazF by induction of inducers and reflected the toxicity of mazF to cells by measuring OD600.


Fig 6. Growth profiles of the engineered bacterium E.coli BL21 in the presence of both inducer IPTG and Au[III], only inducer IPTG or Au[III], and no inducer IPTG and Au[III].


In the presence of both Au[III] and the inducer IPTG, the engineered strains grew normally. Whereas, when only Au[III] or inducer IPTG was present and when neither Au[III] nor inducer IPTG was added, the bacteria expressed the toxic protein mazF resulting in failure to grow normally. The effective operation of the suicide module was demonstrated.