E-COOL-I
Tina Khoury Jeremy Gerbig
Derek BlanchardKerwin 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.
◦ 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.
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
Project’s Original Protocol
Protocol
Isolate biobricks out of well Plates. BBa_I12007 – Promoter Created oligo - RBS BBa_E1010 - Gene BBa_B0015 - Double Terminator
Part 1◦ BBa_I12007
82Bp Promoter: modified lambda Prm Promoter (OR-3 obliterated) 2010 Kit Plate 2 Box 5 Well 11L, pSB2K3
gcaaccattatcaccgccagaggtaaaatagtcaacacgcacggtgttagatatttataaatagtggtgatagatttaacgt
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
Discosoma striata (coral) 2010 Kit Plate 1 Well 18F, pSB2K3
atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa
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
How It Was Suppose To Be Done?
Part 2 RBS to small
Design & Construction of Oligo’s
Transform the bacteria.
Grow the transformed bacteria.
Isolate & check plasmids.◦ Gel Electrophoresis
Protocol Cont.
Ligation of Parts The complete complex Biobricks sequence!
◦ Combine 3 parts BBa_I12007 – Promoter Created Oligo- RBS BBa_E1010 - Gene BBa_B0015 - Double Terminator
Protocol cont… Combining biobrick parts by digestion & ligation.
BBa_I12007 - Promoter BBa_I13503 - RBS + Gene BBa_B0015 - Double Terminator
S X & P X & P
Combining biobrick parts by digestion & ligation.
Protocol cont…
BBa_I12007 –Promoter + Created oligo- RBS BBa_B0015 - Double Terminator
S E & S X & P
BBa_E1010 Gene
Mini-Prep
H2O 5ul
10x FastBuffer
1.5 ul
Enzyme 1 .75ul
Enzyme 2 .75ul
Digestion & Gel Electrophoresis
MasterMix
Ligations
10x ligation Buffer 2ul T4DNA ligase 2ul Gene 1ul Double Terminator 5ul H2O 5ul
Incubate room temp. 1hour
Store at 4℃
BioBrick Part Promoter BBa_I12007 Double terminator BBa_B0015 Ribosome BBa_B0032
Resistance KAN A/K we used KAN AMP
Test tube
A 1ul Promoter part BBa_I12007 1ul DT part BBa_B0015 1ul Ribosome part BBa_B0032
B 5ul Promoter part BBa_I12007 5ul DT part BBa_B0015 5ul Ribosome part BBa_B0032
C 1ul pblue script 1ul pblue script 1ul pblue script
D 1ul H2O 1ul H2O
Bacterial Transformation Results
Test tubes Promoter RBS Double Terminator
A 75 colonies 52 colonies 43 colonies
B 212 colonies 292 colonies 216 colonies
C 1200 colonies
D 0 colonies
• Growth After Plating
Latter 13,12,11,10,9,8,7,6,5, 4, 3,2 1,latter
Isolating of Parts 1st Digestion & Gel
• Our digestion was successful a band of 681 was our target for the Gene
100bp250bp500bp750bp1000bp
100bp250bp500bp750bp1000bp
Gene BBa_E1010 Isolation
Expected Promoter –82bp Double terminator-
129bp
RBS was not ran due to size of only 13bp
Isolating of Parts 10/12
Double Terminator--Promoter
Ladder 1,2, 3,4 5,6,7
100bp250bp500bp750bp1000bp
Double Terminator Promoter
(BBa_B0015) (BBa_I12007)
Expected Gene 681 Bp
1st cut Spe1 Xbal
Redigested◦ Spe1 EcoR1
Redigestion of Gene 10/19
100bp250bp500bp750bp1000bp
1,2,3Gene
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
Ligation Results 11/11
1000bp750bp500bp250bp100bp
15,14,13,12,11,10,9,8,7,6 5,4,3,2,1 ladder
(Promoter & RBS) (Gene & DT)
We Concluded that EcoR1 was not fully functional.
So our parts were not cut open properly to ligate the Gene to the double terminator.
Results 11/16
1000bp750bp500bp250bp100bp
9,8,7,6,5,4,3, 2 1 ladder
Attempted Gene & Double Terminator Ligation
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.
Final Disappointment New Religation of stored Gene & Double Terminator
100bp250bp500bp750bp1000bp
15,14,13,12,11,10,9,8,7,6,5,4,3,2,1 ladder
Attempted Gene & Double Terminator Ligation
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.
Conclusion
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
References