Team:Hong Kong-CUHK/Project/Chloride Sensing Unit
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<li><a class="list-2 selected" href="/Team:Hong_Kong-CUHK/Project/Chloride Sensing Unit">Chloride Sensing Unit</a></li> | <li><a class="list-2 selected" href="/Team:Hong_Kong-CUHK/Project/Chloride Sensing Unit">Chloride Sensing Unit</a></li> | ||
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+ | <img width="600" height="264" src="https://static.igem.org/mediawiki/2011/f/fd/Pgad_operon.jpg" /> | ||
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+ | Fig. 1 P<sub>gad</sub> operon in <em>L. lactis</em>. | ||
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P<sub>gad</sub> is chloride-sensitive promoter which was first discovered in <em>Lactococcuslactis</em><sup>1</sup>, which is a gram-positive bacterium which can live in acidic environment. P<sub>gad</sub> operon (Fig. 1) provides hydrochloric acid feedback mechanism to adjust intracellular metabolism, in order to survive in acidic environment<sup>2</sup>. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of P<sub>gad</sub><sup>2</sup>. The gene before P<sub>gad</sub>, named gadR, is constitutively expressed under the control of P<sub>gadR</sub>, it is a positive regulator of P<sub>gad</sub> coupled genes while intracellular chloride level is elevated<sup>2</sup>. 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 <em>L. lactis</em><sup>2</sup>. | P<sub>gad</sub> is chloride-sensitive promoter which was first discovered in <em>Lactococcuslactis</em><sup>1</sup>, which is a gram-positive bacterium which can live in acidic environment. P<sub>gad</sub> operon (Fig. 1) provides hydrochloric acid feedback mechanism to adjust intracellular metabolism, in order to survive in acidic environment<sup>2</sup>. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of P<sub>gad</sub><sup>2</sup>. The gene before P<sub>gad</sub>, named gadR, is constitutively expressed under the control of P<sub>gadR</sub>, it is a positive regulator of P<sub>gad</sub> coupled genes while intracellular chloride level is elevated<sup>2</sup>. 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 <em>L. lactis</em><sup>2</sup>. | ||
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- | J. Sanders et al. tried to develop chloride-sensitive expression cassette using P<sub>gad</sub> operon<sup>3</sup>. They constructed the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes P<sub>gadR</sub>, gadR, P<sub>gad</sub> and the starting codon ATG, and replaced downstream report genes<sup>3</sup>. They | + | J. Sanders et al. tried to develop chloride-sensitive expression cassette using P<sub>gad</sub> operon<sup>3</sup>. They constructed the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes P<sub>gadR</sub>, gadR, P<sub>gad</sub> and the starting codon ATG, and replaced downstream report genes<sup>3</sup>. They succeeded in transforming the cassette to <em>E.coli</em> and varying the expression of target genes under different sodium chloride concentrations<sup>3</sup>. In our project, we try to build light-coupled chloride expression switch based on this design. |
</p> | </p> | ||
+ | <p>If you are insterested in our result on this part, you might visit the "<a href="https://2011.igem.org/Team:Hong_Kong-CUHK/Project/light">Light Intra-tunable system</a>" session.</p> | ||
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References | References | ||
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3. Sanders, J.W., Venema, G. & Kok, J. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. <em>Appliedand environmental microbiology</em> <strong>63</strong>, 4877(1997). | 3. Sanders, J.W., Venema, G. & Kok, J. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. <em>Appliedand environmental microbiology</em> <strong>63</strong>, 4877(1997). | ||
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Latest revision as of 18:51, 28 October 2011
Chloride Sensing Unit
![](https://static.igem.org/mediawiki/2011/f/fd/Pgad_operon.jpg)
Fig. 1 Pgad operon in L. lactis.
Pgad is chloride-sensitive promoter which was first discovered in Lactococcuslactis1, which is a gram-positive bacterium which can live in acidic environment. Pgad 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 glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, is constitutively expressed under the control of PgadR, it is a positive regulator of Pgad coupled genes while intracellular chloride level is 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 develop chloride-sensitive expression cassette using Pgad operon3. They constructed the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG, and replaced downstream report genes3. They succeeded in transforming the cassette to E.coli and varying the expression of target genes under different sodium chloride concentrations3. In our project, we try to build light-coupled chloride expression switch based on this design.
If you are insterested in our result on this part, you might visit the "Light Intra-tunable system" session.
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).
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