ampicillin resistant native promoter wild type enzyme 5’5’3’3’ hindiii start codon stop...

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

pASK IBA 43+