Difference between revisions of "Part:BBa K594002:Design"

Latest revision as of 03:26, 6 October 2011

subunit of a 3-isopropylmalate dehydrogenase from E.coli BL21

Design Notes

Besides, when we were looking for leuB gene, we also try to find its RBS, but unluckily, we didn't find its RBS on NCBI, its location is closely linked to leuA's location.

The verification

leuB is a necessary part in leu operon.its metabolic mechanism is like this picture.

We have sent the sinI to the sequencing center with primer VR, VF2, and it proves that it is 100% correct.

the sequecing result,

AAATCTTGACCTTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGA
TTTCTGGAATTCGCGGCCGCTTCTAGATGTCGAAGAATTACCATATTGCCGTATTGCCGGGGGACGGTATTGGTCCGGAAGTGAT
GACCCAGGCGCTGAAAGTGCTGGATGCCGTGCGCAACCGCTTTGCGATGCGCATCACCACCAGCCATTACGATGTAGGCGGCGCA
GCCATTGATAACCACGGGCAACCACTGCCGCCTGCGACGGTTGAAGGTTGTGAGCAAGCCGATGCCGTGCTGTTTGGCTCGGTAG
GCGGCCCGAAGTGGGAACATTTACCACCAGACCAGCAACCAGAACGCGGCGCGCTGCTGCCTCTGCGTAAGCACTTCAAATTATT
CAGCAACCTGCGCCCGGCAAAACTGTATCAGGGGCTGGAAGCATTCTGTCCGCTGCGTGCAGACATTGCCGCAAACGGCTTCGAC
ATCCTGTGTGTGCGCGAACTGACCGGCGGCATCTATTTCGGTCAGCCAAAAGGCCGCGAAGGTAGCGGACAATATGAAAAAGCCT
TTGATACCGAGGTGTATCACCGTTTTGAGATCGAACGTATCGCCCGCATCGCGTTTGAATCTGCTCGCAAGCGTCGCCACAAAGT
GACGTCGATCGATAAAGCCAACGTGCTGCAATCCTCTATTTTATGGCGGGAGATCGTTAACGAGATCGCCACGGAATACCCGGAT
GTCGAACTGGCGCATATGTACATCGACAACGCCACCATGCAGCTGATTAAAGATCCATCACAGTTTGACGTTCTGCTGTGCTCCA
ACCTGTTTGGCGACATTCTGTCTGACGAGTGCGCAATGATCACTGGCTCGATGGGGATGTTGCCTTCCGCCAGCCTGAACGAGCA
AGGTTTTGGACTGTATGAACCGGCGGGCGGCTCGGCACCAGATATCGCAGGCAAAAACATCGCCAACCCGATTGCACAAATCCTT
TCGCTGGCACTGCTGCTGCGTTACAGCCTGGATGCCGATGATGCGGCTTGCGCCATTGAACGCGCCATTAACCGCGCATTAGAAG
AAGGCATTCGCACCGGGGATTTAGCCCGTGGCGCTGCCGCCGTTAGTACCGATGAAATGGGCGATATCATTGCCCGCTATGTAGC
AGAAGGGGTGTAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACC
GAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTCCGCTGCGCAGCCCCCG

We use NCBI blast to analysis the sequence, finally get the result.

Score = 2017 bits (1092), Expect = 0.0, Identities = 1092/1092 (100%), Gaps = 0/1092 (0%) Strand=Plus/Plus

So the leuB is right, when added proper promoter and RBS, it will work,

Source

This leuB is from the leu operon in BL 21(E.coli),we get BL21 from Takara and found its sequence in NCBI, then cloned it with a Pfu PCR.

References

1.J. M. Somers, A. Amzallag1, and R. B. Middleton2，Genetic Fine Structure of the Leucine Operon of Escherichia coli K-12，J Bacteriol. 1973 March; 113(3): 1268-1272
2.Wallon G, Yamamoto K, Kirino H, Yamagishi A, Lovett ST, Petsko GA, Oshima T (1997). "Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli." Biochim Biophys Acta 1337(1);105-12. PMID: 9003442
3. Wallon G, Kryger G, Lovett ST, Oshima T, Ringe D, Petsko GA (1997). "Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus." J Mol Biol 266(5);1016-31. PMID: 9086278
4.Wright BE, Longacre A, Reimers JM (1999). "Hypermutation in derepressed operons of Escherichia coli K12." Proc Natl Acad Sci U S A 96(9);5089-94. PMID: 10220423
5. Yang HL, Kessler DP (1974). "Genetic analysis of the leucine region in Escherichia coli B-r: gene-enzyme assignments." J Bacteriol 117(1);63-72. PMID: 4587614