ampicillin resistant native promoter wild type enzyme 5’5’3’3’ hindiii start codon stop...
TRANSCRIPT
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Ampicillin resistantNative promoter
Wild type enzyme
5’ 3’
HindIII
StartCodon
StopCodon
BsteII
phoA in pEK8
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Potential New Plasmids
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pET29a5’ 3’
HindIII
StartCodon
StopCodon
BsteII
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pET29a
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We want a C-terminal histidine tagWe do not want an S-Tag
5’ 3’
HindIII
StartCodon
StopCodon
BsteII
![Page 6: Ampicillin resistant Native promoter Wild type enzyme 5’5’3’3’ HindIII Start Codon Stop Codon BsteII phoA in pEK8](https://reader036.vdocuments.mx/reader036/viewer/2022062309/5697bfe31a28abf838cb5432/html5/thumbnails/6.jpg)
We want a C-terminal histidine tagWe do not want an S-Tag
New TAAStop codon
5’ 3’
HindIII
StartCodon
StopCodon
BsteII
5’ 3’
HindIII
StartCodon
StopCodon
BsteIINdeI XhoI
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phoA Primer Design
•Find the sequence of phoA in pEK48
•Append new endonuclease cut sites
•Delete the stop codon
•Do some tinkering with Tm and ends
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phoA SequenceExpasy Translate
TTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTGCACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAAAAAAACCGGCAAACCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCAAAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAATTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCA
GTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA
E. coli strain K-12 bases 400902 - 402368
NCBI BLAST
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E. coli K-12 Start Codons
•4288 open reading frames
•3542 start with ATG, 83% methionine
•612 start with GTG, 14% valine
•130 start with TTG, 3% luecine
Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5 277(5331):1453-62
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Forward PrimerNdeI
5’ TTGAUGTCACGGCCGAGACTTATAGTCGCTTTG
5’ CATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG
5’ AAAAAAAACATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG
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Reverse PrimerXhoI
Reverse and complement
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAG
5’AAAAAAAACTCGAGTTTCAGCCCCAGAGCGGCTTTCATGGTGTAGAAGAGATCGGT
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAGTTTTTTTT
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Melting Temperature
•Melting temperature to be between 55 and 60 C
•Melting temperatures to be as close to one another as possible
Melting Temperature Calculator