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E-COOL-I

E-COOL-I. Tina Khoury Jeremy Gerbig Derek Blanchard Kerwin Dunham. Original 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.

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E-COOL-I

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  1. E-COOL-I Tina Khoury Jeremy Gerbig Derek Blanchard Kerwin Dunham

  2. Original 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. Project’s Original Protocol • Isolate plasmid • Digest with appropriate enzymes. • Confirm base pair length • Ligation of confirmed digested Biobrick parts • Ligate final Biobrick arrangement • Confirm arrangement and biobrick standards • Grow under different environmental conditions

  4. Protocol • Isolate biobricks out of well Plates. • BBa_I12007 – Promoter • Created oligo - RBS • BBa_E1010 - Gene • BBa_B0015 - Double Terminator

  5. How It Was Suppose To Be Done? • Part 1 • BBa_I12007 • 82Bp • Promoter: modified lambda Prm Promoter • (OR-3 obliterated) • 2010 Kit Plate 2 Box 5 Well 11L, pSB2K3 • gcaaccattatcaccgccagaggtaaaatagtcaacacgcacggtgttagatatttataaatagtggtgatagatttaacgt

  6. How It Was Suppose To Be Done? • Part 3 Gene • Spring 2008 Distribution Source Plate 1002 1D pSB1A2 • 3 BBa_E1010 • 681Bp • Gene: highly engineered mutant of red fluorescent protein from Discosomastriata (coral) • 2010 Kit Plate 1 Well 18F, pSB2K3 • atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

  7. How It Was Suppose To Be Done? • 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

  8. Design & Construction of Oligo’s • Part 2 • RBS to small

  9. Protocol Cont. • Transform the bacteria. • Grow the transformed bacteria. • Isolate & check plasmids. • Gel Electrophoresis

  10. Ligation of Parts • The complete complex Biobricks sequence! • Combine 3 parts • BBa_I12007 – Promoter • Created Oligo- RBS • BBa_E1010 - Gene • BBa_B0015 - Double Terminator

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

  12. Protocol cont… • Combining biobrick parts by digestion & ligation. • BBa_I12007 –Promoter + • Created oligo- RBS • BBa_E1010 Gene • BBa_B0015 - Double Terminator E & S X & P S

  13. Mini-Prep

  14. Digestion & Gel Electrophoresis MasterMix

  15. Ligations • 10x ligation Buffer 2ul • T4DNA ligase 2ul • Gene 1ul • Double Terminator 5ul • H2O 5ul • Incubate room temp. 1hour • Store at 4℃

  16. Bacterial Transformation Results • Growth After Plating

  17. Isolating of Parts 1st Digestion & Gel • Our digestion was successful a band of 681 was our target for the Gene Latter 13,12,11,10,9,8,7,6,5, 4, 3,2 1,latter • 100bp • 250bp • 500bp • 750bp • 1000bp • 100bp • 250bp • 500bp • 750bp • 1000bp Gene BBa_E1010 Isolation

  18. Isolating of Parts 10/12 Expected • Promoter –82bp • Double terminator-129bp • RBS was not ran due to size of only 13bp • 100bp • 250bp • 500bp • 750bp • 1000bp Ladder 1,2, 3,4 5,6,7 Double Terminator Promoter (BBa_B0015) (BBa_I12007) Double Terminator--Promoter

  19. Redigestion of Gene 10/19 • Expected Gene 681 Bp • 1st cut Spe1 Xbal • Redigested • Spe1 EcoR1 • 100bp • 250bp • 500bp • 750bp • 1000bp • 1,2,3 • Gene

  20. Ligation Results 11/11 (Promoter & RBS) (Gene & DT) • Ligation problem possibly low functioning EcoR1 • Promoter & RBS – not sure if ligated correctly due to RBS small size 13bp. Difference can’t be seen on gel • Gene & DT – Believe only DT is showing up 129bp 15,14,13,12,11,10,9,8,7,6 5,4,3,2,1 ladder • 1000bp • 750bp • 500bp • 250bp • 100bp

  21. Results 11/16 • We Concluded that EcoR1 was not fully functional. • So our parts were not cut open properly to ligate the Gene to the double terminator. 9,8,7,6,5,4,3, 2 1 ladder • 1000bp • 750bp • 500bp • 250bp • 100bp Attempted Gene & Double Terminator Ligation

  22. Final Disappointment New Religation of stored Gene & Double Terminator • No proper digestions around 810bp. • No Ligation? • Error Analysis: • Still low functioning enzymes & Resuspension fluid had been left out of 4C for undisclosed amount of time. 15,14,13,12,11,10,9,8,7,6,5,4,3,2,1 ladder • 100bp • 250bp • 500bp • 750bp • 1000bp Attempted Gene & Double Terminator Ligation

  23. Conclusion We were successful in isolating and confirming 3 of our Biobrick parts. Gene Promoter Double terminator. Given more time, fresh enzymes and other properly working materials ligating of our biobrick parts would have been successful.

  24. 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 • http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/images/bacterial_transformation/4063-1-eng-AU/bacterial_transformation_large.jpg

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