Team:Hong Kong-CUHK/Project/Chloride Sensing Unit

Background

P_gad is chloride-sensitive promoter which was first discovered in Lactococcuslactis¹, which is a gram-positive bacterium which can live in acidic environment. P_gad operon (Fig. 1) provides hydrochloric acid feedback mechanism to adjust intracellular metabolism, in order to survive in acidic environment². In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamatedecarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of P_gad². The gene before P_gad, named gadR, which is constitutively expressed under the control of P_gadR, is a positive regulator of P_gad coupled genes while intracellular chloride is level elevated². When intracellular pH decreases, the expression of gadB and gadC is enhanced due to the action of gadR and confers glutamate-dependent acid resistance in L. lactis².

J. Sanders et al. tried to developchloride-sensitive expression cassette using P_gad operon³.  They constructed the cassette from bp 821 to2071 of GenBank sequence AF005098, which includes P_gadR, gadR, P_gadand the starting codon ATG, and replaced downstream report genes³. They managetransforming the cassette to E.coli and varying the expression of report genes under different sodium chloride concentrations³. In our project, we try to build light-coupled chloride expression switch based on this design.

References

1.        Sanders, J.W. et al. Identifcation of a sodium chloride-regulated promoter in Lactococcus lactis by single-copy chromosomal fusion with a reporter gene. Mol Gen Genet 257, 681-685(1998).

2.        Sanders, J.W. et al. A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation. Molecular microbiology 27, 299-310(1998).

3.        Sanders, J.W., Venema, G. & Kok, J. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. Appliedand environmental microbiology 63, 4877(1997).