1 / 13

phoA in pEK8

HindIII. BsteII. Start Codon. Stop Codon. 5 ’. 3 ’. phoA in pEK8. Ampicillin resistant Native promoter Wild type enzyme. Potential New Plasmids. HindIII. BsteII. Start Codon. Stop Codon. 5 ’. 3 ’. pET29a. pET29a. Start Codon. Stop Codon.

alissa
Download Presentation

phoA in pEK8

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. Potential New Plasmids

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

  4. pET29a

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

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

  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 NCBI BLAST Expasy Translate TTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTGCACCCGGCGGTGCTCGCCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGTAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATAGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAAAAAAACCGGCAAACCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACCGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCACATGTGACCTCGCGCAAATGCTACGGTCCGAGCGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCTCGTGCCGACGTTACGCTTGGCGGCGGCGCAAAAACCTTTGCTGAAACGGCAACCGCTGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATGCTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCAAAAACCCCTGCTTGGCCTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCTAGGACCGAAAGCAACGTACCATGGCAATATCGATAAGCCCGCAGTCACCTGTACGCCAAATCCGCAACGTAATGACAGTGTACCAACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCATGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAATTCGCTAAAAAGGAGGGTAACACGCTGGTCATAGTCACCGCTGATCACGCCCACGCCAGCCAGATTGTTGCGCCGGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCAGTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA E. coli strain K-12 bases 400902 - 402368

  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’AAAAAAAACATATGAUGTCACGGCCGAGACTTATAGTCGCTTTG

  11. Reverse Primer XhoI 5’ ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAATAA 5’ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAG 5’ACCGATCTCTTCTACACCATGAAAGCCGCTCTGGGGCTGAAACTCGAGTTTTTTTT Reverse and complement 5’AAAAAAAACTCGAGTTTCAGCCCCAGAGCGGCTTTCATGGTGTAGAAGAGATCGGT

  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+

More Related