ampicillin resistant native promoter wild type enzyme 5’5’3’3’ hindiii start codon stop...
TRANSCRIPT
Ampicillin resistantNative promoter
Wild type enzyme
5’ 3’
HindIII
StartCodon
StopCodon
BsteII
phoA in pEK8
Potential New Plasmids
pET29a5’ 3’
HindIII
StartCodon
StopCodon
BsteII
pET29a
We want a C-terminal histidine tagWe do not want an S-Tag
5’ 3’
HindIII
StartCodon
StopCodon
BsteII
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
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
phoA SequenceExpasy Translate
TTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTGCACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAAAAAAACCGGCAAACCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCAAAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAATTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCA
GTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA
E. coli strain K-12 bases 400902 - 402368
NCBI BLAST
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
Forward PrimerNdeI
5’ TTGAUGTCACGGCCGAGACTTATAGTCGCTTTG
5’ CATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG
5’ AAAAAAAACATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG
Reverse PrimerXhoI
Reverse and complement
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAG
5’AAAAAAAACTCGAGTTTCAGCCCCAGAGCGGCTTTCATGGTGTAGAAGAGATCGGT
5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAGTTTTTTTT
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
