Revision as of 13:14, 21 October 2019

lldRO1-PfnrF8-lldRO2(Phll), response to high lactate and hypoxia

This is a composite part consists of lldRO1-J23117-lldRO2 (<ahref="https://parts.igem.org/Part:BBa_K1847007">BBa_K1847007</a>) and PfnrF8 (<ahref="https://parts.igem.org/Part:BBa_K2913009">BBa_K2913009</a>). It has a functional improvement of lldRO1-J23117-lldRO2 via tansforming a specific Anderson promoter (<a href="https://parts.igem.org/Part:BBa_J23117">BBa_J23117</a>) to a hypoxia-inducible promoter, PfnrF8. The lldRO1--lldRO2 was utilized with other parts of the lldPRD operon (previously named as lct), responsible for aerobic L-lactate metabolism. The lldPRD operon consists of three genes that form a single transcriptional unit inducible by growth in L-lactate. The three genes lldD, lldP and lldR encode a dehydrogenase, a permease and a regulatory protein, respectively. We chose lldR and lldP to assist building our lactic acid response unit. lldR protein can bind to the operators O1 and O2 located on each side of the PfnrF8. When lactic acid level is low, two lldR molecules will individually bind to the O1 and O2 sites and form a tetramer to make DNA strand form a hairpin structure, which can turn off the expression of the downstream gene. When lactic acid level reaches a certain point, lldR will be released from the O1 and O2 operators, and the DNA hairpin will be resolved, leading to activated transcription of the downstream gene. We also used lldP to increase the sensitivity of our system to lactic acid alteration.

Usage and Biology

result

We compared the response of the Phll to hypoxia and normal oxygen environments after adding sodium lactate of different concentrations (0, 10, 25, 50, 200, 300 mM). (fig.1b) We thought that β-galactosidase expression is higher when sodium lactate and sodium sulfite absent in the medium because the growth condition of bacteria is better than that of bacteria cultured in the medium with sodium lactate and sodium sulfite. Under the conditions of hypoxia and high lactic acid, the expression of β-galactosidase was significantly increased, which indicated that our Phll was effective.

Fig.1(a) In an environment of normal oxygen and low lactic acid, two lldR molecules will form a homo-tetramer, and the binding of the transcription factor FNR to the hypoxia-inducible promoter will be impeded, leading to expressing suppression of the downstream gene. In an environment of hypoxia and high lactic acid level, lldR will be released from the O₂ site and the transcription factor can bind to the PfnrF8 promoter, leading to the expression of downstream gene. (b) LacZ expression in E. coli Nissle 1917 was induced by different concentrations of sodium lactate (0mM, 10mM, 25mM, 50mM, 200mM and 300mM) with (+) or without (-) 1 g/l Na₂SO₃. β-gal activity was measured as above.

References

[1] Wu, Y., et al., Lactate, a Neglected Factor for Diabetes and Cancer Interaction. Mediators Inflamm, 2016. 2016: p. 6456018.

[2] Goers, L., et al., Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng, 2017. 114(6): p. 1290-1300.

[3] Aguilera, L., et al., Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol, 2008. 190(8): p. 2997-3005.

Sequence and Features