Part:BBa_K2913023

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

This is a composite part consists of lldRO1-J23117-lldRO2 BBa_K1847007 and PfnrF8 BBa_K2913009. It has a functional improvement of lldRO1-J23117-lldRO2 via tansforming a specific Anderson promoter BBa_J23117 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.

Method

To test the effectiveness of the promoter that responds to both hypoxic and high lactate signals (Phll) we created, we provided conditions that were low in oxygen and high in lactic acid to see if the element worked. We put the lacZ gene downstream the Phll regulating promoter to select by ONPG detection of β-galactosidase activity. In order to prevent the growth inhibition of bacteria by excessively low pH, we use sodium lactate instead of lactic acid for induction. We use sodium sulfite (1 g/l Na₂SO₃) and a series of concentrations of sodium lactate (10, 25, 50, 200 and 300 mM) to create hypoxia and high lactic acid environment. Culture bacteria for 8-12 h at 16 ℃ in a shaker. Record the fluorescence value at 420nm for each well every 30 seconds in an automatic microplate reader, 37 ℃, 1-2 h. Each group should be repeated for at least 3 times.

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 (0, 10, 25, 50, 200, 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

Assembly Compatibility:

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

  Illegal NheI site found at 92
  Illegal NheI site found at 115
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]