1. Ampicillin resistant Native promoter Wild type enzyme 5’5’3’3’ HindIII Start Codon Stop Codon BsteII phoA in pEK8

2. Potential New Plasmids

3. pET29a 5’5’3’3’ HindIII Start Codon Stop Codon BsteII

4. pET29a

5. We want a C-terminal histidine tag We do not want an S-Tag 5’5’ 3’3’ HindIII Start Codon Stop Codon BsteII

6. We want a C-terminal histidine tag We do not want an S-Tag New TAA Stop codon 5’5’ 3’3’ HindIII Start Codon Stop Codon BsteII 5’5’ 3’3’ HindIII Start Codon Stop Codon BsteII NdeIXhoI

7. 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

8. phoA Sequence Expasy Translate TTGTCACGGCCGAGACTTATAGTCGCTTTG TTTTTATTTTTTAATGTATTTGTACATGGAGAAAATAAAGTGAAACAAAGCAC TATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTAC TGCACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGAT GGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACT CACTATGCGCTGAATAAAAAAACCGGCAAACCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGC GCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAG GATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAA GGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTG AATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCA AAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCT GTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGC AAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTG GAATTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCA CCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCAGTTGCGTATTGCG GCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAG ACCGATCTCTTCTACACCATGAAAGCCGCTCT GGGGCTGAAATAA E. coli strain K-12 bases 400902 - 402368 NCBI BLAST

9. 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

10. Forward Primer NdeI 5’ TTGAUGTCACGGCCGAGACTTATAGTCGCTTTG 5’ CATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG 5’ AAAAAAAACATATGAUGTCACGGCCGAGACTTATAGTCGCTT TG

11. Reverse Primer XhoI Reverse and complement 5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATA A 5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAG 5’AAAAAAAACTCGAGTTTCAGCCCCAGAGCGGCTTTCATGGTGTAGAAGAGATCGGT 5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAGTTTT TTTT

12. 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

13. pASK IBA 43+