Team:IIT Delhi/Biobrick

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BBa_K1170000

Acid Shock Response (asr) promoter is an acid-inducible promoter sequence, present in the genome of E. coli K12 bacteria.



Fig 1: Position of acid shock response (asr) gene in the genome of E. coli K12. +1 site denotes the Transcriptional Start Site of the asr gene, -10 denotes the TATA box and the “Pho Box” denotes the TF site that is regulated by “Pho operon” in E. coli.


When performed in LPM (Low Phosphate Minimal) media, induction due to asr promoter is almost 100 times at it’s most optimal pH, i.e. 4.8, as compared to pH 7.0 [1].



Fig 2: Northern Blot Analysis of the RNA isolated from E. coli K12 strain induction at two different pH at multiple time intervals, indicates significant production of mRNA transcribed under the P-asr promoter.


We identified a minimal promoter region of 178bp, just before the ORF of the asr gene, that included the observable regulatory regions for acid induction.

CGATCAAGACTACTATTATTGGTAGCTAAATTTCCCTTAAGTCACAATACGTTATTATCAACGCTGTAATTTATTCAGCGTTTGTACATAT
CGTTACACGCTGAAACCAACCACTCACGGAAGTCTGCCATTCCCAGGGATATAGTTATTTCAACGGCCCCGCAGTGGGGTTAAATGA

We designed the following primers for this sequence and PCR amplified the fragment from E. coli K12 genome (these are not in Standard Biobrick configuration as, initially, we used pUC19 as our vector for cloning):

FP: GTCGAATTCCGATCAAGACTACTATTATTGGTAGCT (36) (EcoRI site)
RP: GTAGGATCCTCATTTAACCCCACTGCGGGGCCGTTGAAATAAC (43) (BamHI site)


BBa_K1170001

Following is the Super Folder Green Fluorescent Protein (SFGFP) encoding DNA sequence submitted to the registry:

ATGCGTAAAGGCGAGGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTG
GCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGT
AACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGC
TATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACC
GCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACAT
CACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTAC
CAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACG
AGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATGATGA
(GenBank ID: HQ873313.1 GI: 321437460)

This sequence is almost identical to that of BBa_I746916 (Designed by iGEM 2007 Team from Cambridge). However, on a quick BLAST alignment of the two sequences, we observe that there is a mutation at the 15th bp, making it a novel part in the registry.

Following primers were designed for this part, for cloning it under P-asr in pUC19 plasmid:
FP: GTAGGATCCATGCGTAAAGGCGAGGAGCTGTTCACTGGT (39) (BamHI)
RP: GTACTGCAGTCATCATTTGTACAGTTCATCCATACCATG (39) (PstI)
(Again, these are different from the standard Biobrick parts, as required by iGEM).


BBa_K1170002

This is a reporter gene that we planned to use in our pH sensor for detecting alkaline surroundings. LacZ gene, present in the ORF of the lac operon in E. coli, encodes for β-Galactosidase enzyme which hydrolyses the β-(1,4) bond between a glucose unit (α or β form) and a galactose unit (only β form). This intracellular enzyme can be used to react with X-Gal as substrate and produce a blue colored dye to act as a visible reporter of lacZ gene expression. Since our aim is to engineer bacteria so that it behaves akin to a Universal pH indicator, lacZ gene is ideal for expression under an alkali-inducible promoter and generate blue color at high pH. The sequence submitted to the iGEM registry is as follows:

>BBa_K1170002 Part-only sequence (3018 bp)

atgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacat
ccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcc
tggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatg
cacggttacgatgcgcccatctacaccaacgtaacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttac
tcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgc
aacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcg
gtgatggtgctgcgttggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcat
aaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgc
ggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaatt
atcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctat
cgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggt
ctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacg
atggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgc
gaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggcta
ccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccac
ggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggcc
accgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgcta
cctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgt
cagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttac
ggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaa
gcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgag
ctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaa
ctgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccggg
cacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagc
gaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaa
caactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcc
tgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgatt
acgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattacc
gttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctc
ggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtac
gtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagc
cgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatg
gggattggtggcgacgactcctggagcccgtcagtatcggcg [GI: J01636.1]


BBa_K1170003

The 733bp Alkali-inducible promoter (P-atp2), present in the F¬¬0F1 ATPase operon in Corynebacterium glutamicum ATCC 13032, can be used to elicit a high pH-based response [2].



Fig: a) Organization of the F0F1 ATPase operon in C. glutamicum. Note that the operon apparently has two overlapping promoters, P-atp1 regulating the 1.2kb fragment (not well characterized alkali-induction) and P-atp2 inducing the 7.5kb fragment at high pH. b) Northern Hybridization analysis of alkali induction of the F0F1 operon in C. glutamicum.






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References:
[1] Suziedeliené, E., Suziedélis, K., Garbenciūté, V., Normark, S. The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon. J. Bacteriol. (1999)
[2] Mo´nica Barriuso-Iglesias, Carlos Barreiro, Fabio Flechoso and Juan F. Martı´n, Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH. Microbiology January 2006 152:1 11-21