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E. Coli Fluorescing Red at Cold Temperature Sensor

E. Coli Fluorescing Red at Cold Temperature Sensor. Prm + I12007. . B0032. Team: E Cool I Tina Khoury Jeremy Gerbig Kerwin Dunham Derek Blanchard. Goals. Achieve E. coli to fluoresce red at low temp (37°C) in presence of Cl or Cl (ts).

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E. Coli Fluorescing Red at Cold Temperature Sensor

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  1. E. Coli Fluorescing Red at Cold Temperature Sensor Prm + I12007 . B0032 Team: E Cool I Tina Khoury Jeremy Gerbig Kerwin Dunham Derek Blanchard

  2. Goals • Achieve • E. coli to fluoresce red at low temp (37°C) in presence of Cl or Cl (ts). • Find optimum temp where color change will be found. • ~ 30-37°C • Find optimum concentration of Cl. • Gene originally from coral. • Backup Plan • Use high temp parts to make E. coli fluoresce at high temp instead at low using a different gene. • Expressing high (green) and low (red) temp. genes in one sequence.

  3. How to do it? • Part 1 • BBa_I12007 • 82Bp • Promoter: modified lambda Prm Promoter • (OR-3 obliterated) • 2010 Kit Plate 2 Box 5 Well 11L, pSB2K3 • gcaaccattatcaccgccagaggtaaaatagtcaacacgcacggtgttagatatttataaatagtggtgatagatttaacgt

  4. Part 2 & 3 Super Part BBa_I13503 • Spring 2008 Distribution Source Plate 1002 1D pSB1A2 • BBa_B0032 • 13Bp • Ribosome Binding Site RSB.3 • (medium)- derivative of BBa_0030 • 2010 Kit Plate 1 Well 2I, pSB1A2 • tcacacaggaaag

  5. Part 2 & 3 Super Part BBa_I13503 • Spring 2008 Distribution Source Plate 1002 1D pSB1A2 • 3 BBa_E1010 • 681Bp • Gene: highly engineered mutant of red fluorescent protein from Discosoma striata (coral) • 2010 Kit Plate 1 Well 18F, pSB2K3 • atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

  6. Part 4 & 5 Super Part BBa_B0015 • BBa_B0010 doubleT • 129 Bp • Stop, T1 from E. coli rrn B • (Transcriptional Terminator) • 2010 Kit Plate 1 Well 13D, pSB1A2 • BBa_B0012 • Stop, TE from coliophage T7 • (Transcriptional Terminator) • Source Plate 1000 Well 1B, pSB1A2 • ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

  7. What’s new? • The complete complex Biobricks sequence that works! • Combine 3 parts • BBa_I12007 - Promoter • BBa_I13503 - RBS + Gene • BBa_B0015 - Double Terminator Promoter RBS + Gene Double Terminator

  8. Alternate Part • May use double terminator because the first terminator has had problems working according to partsregistry.org • Possible double terminator is BBa_B0015 double terminator(B0010-B0012) 2010 Kit Plate 1 Well 23L, pSB1AK3. According to the website, this part works well.

  9. Protocol • Isolate biobricks out of wells. • BBa_I12007 - Promoter • BBa_I13503 - RBS + Gene • BBa_B0015 - Double Terminator • Transform the bacteria. • Grow the transformed bacteria. • Isolate & check plasmids. • Gel Electrophoresis

  10. Protocol cont… • Combining biobrick parts by digestion & ligation. • BBa_I12007 - Promoter • BBa_I13503 - RBS + Gene • BBa_B0015 - Double Terminator S X & P X & P

  11. Protocol cont…. • Transform bacteria with new recombinant plasmid. • Observe results • Color change dependent on • Temp between ~ 30-37°C • Cl concentration ~ 1x – 10x

  12. References • Openwetware.org • Partsregistry.org • http://filebox.vt.edu/.../biol_4684/Methods/genes.html • http://www.fasebj.org/content/vol20/issue14/images/large/z386120661480003.jpeg • http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mga&part=A1549 • http://www.stat.berkeley.edu/users/terry/Classes/s260.1998/Week8b/week8b/node3.html

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