Team:NYMU-Taipei/Project/pH Sensor

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pH Sensor

Experiments and Parts

About Us

Motivation

When our system arrives in the intestine (pH is higher), it senses the pH change and starts to work.
In this subsystem, we are going to create a pH sensor which senses high pH's.

Goal

The pH sensor can sense a high pH condition and start gene expression.

Circuit Design

Regulation of nhaA gene expression

[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=178537&blobtype=pdf Na1-Induced Transcription of nhaA, Which Encodes an Na1/H1 Antiporter in Escherichia coli, Is Positively Regulated by nhaR and Affected by hns] (N. DOVER etc.,1996)
[http://jeb.biologists.org/cgi/content/abstract/196/1/443 Molecular physiology of the Na+/H+ antiporter in Escherichia coli] (E Padan and S Schuldiner, 1994)

Structure of nhaA gene promoter

[http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11133959 Transcription of nhaA, the Main Na+/H+ Antiporter of Escherichia coli, Is Regulated by Na+ and Growth Phase] (Nir Dover and Etana Padan et al.,2001)

17,189 -17,488 in E.coli K12 genome from Ecocyc (300 bp upstream of nhaA gene)


00000000011111111112222222222344444444455555555556666666666777777777788888888889
12345678901234567890123456789012345678901234567890123456789012345678901234567890
ACGACAAGCTGGATTATTTTTGAAATATTGGCCTAACAAGCATCGCCGACTGACAACAAATTAATTATTACTTTTCCTAA
TTAATCCCTCAGGAATCCTCACCTTAAGCTATGATTATCTAGGCTTAGGGTCACTCGTGAGCGCTTACAGCCGTCAAAAA
CGCATCTCACCGCTGATGGCGCAAATTCTTCAATAGCTCGTAAAAAACGAATTATTCCTACACTATAATCTGATTTTAAC
GATGATTCGTGCGGGGTAAAATAGTAAAAACGATCTATTCACCTGAAAGAGAAATAAAAA

G is the start of NhaR binding site
TTTTAA is the first -35 of P1
ATGATT is the second -35 of P1
TAAAAT is the first -10 of P1
TAAAAA is the second -10 of P1; A in the TAAAAA is the first TSS of P1
AAGAGA is the S.D. (Shine-Dalgarno) sequence in RBS
There are 3 nhaA promoter sequences protected by NhaR from DNase I digestion
- AATAGCTCGTAAAAAACGAATTATTCC
- CACTATAATCTGATTTTAACGATG
- CGTGCGGGGTAAAATAGTAAAAACGATCTATTCACCT; T in TTCACCT is the second TSS of P1
The extracted DNA sequence should include the NhaR binding site
- The NhaR binding site defined in (Nir Dover and Etana Padan et al.,2001) is 120 bp long
- The PCR product derived from primers designed by Henry is 274 bp long

References

[http://jeb.biologists.org/cgi/content/abstract/196/1/443 Molecular physiology of the Na+/H+ antiporter in Escherichia coli] (E Padan and S Schuldiner, 1994)
[http://www.pnas.org/cgi/content/abstract/90/4/1212 Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli] (Y Gerchman etc., 1993)
[http://www.nature.com/nature/journal/v435/n7046/full/nature03692.html Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH] (Carola Hunte etc., 2005)
[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=178537&blobtype=pdf Na1-Induced Transcription of nhaA, Which Encodes an Na1/H1 Antiporter in Escherichia coli, Is Positively Regulated by nhaR and Affected by hns] (N. DOVER etc.,1996)
[http://jeb.biologists.org/cgi/content/abstract/196/1/443 Molecular physiology of the Na+/H+ antiporter in Escherichia coli] (E Padan and S Schuldiner, 1994)
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1S-4CVR4C8-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5cb90ff8555f4439c804a59f5b397c16 NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter] (E. Padan etc., 2004)
[http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11133959 Transcription of nhaA, the Main Na+/H+ Antiporter of Escherichia coli, Is Regulated by Na+ and Growth Phase] (Nir Dover and Etana Padan et al.,2001)