IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week0

=Monday: June 16, 2008=

Basic Idea

 * Use a chemical signal to induce electric output
 * Use E. coli as detector, Shewie as indicator
 * Use E. coli to produce lactate, needed for e- production in Shewie

Logistics of Game
Person v. Person 
 * Example game:
 * 1) Player 1 adds chemical
 * 2) Computer tracks electric output and displays move on screen
 * 3) Player 2 adds chemical
 * 4) Computer tracks electric output and displays move on screen
 * 5) Repeat
 * One chemical detection needed

Person v. Computer
 * Example game:
 * 1) Player 1 adds chemical
 * 2) Computer tracks electric output and displays move on screen
 * 3) Computer chooses its move and lights up the well that it decides to move to
 * 4) Bacteria respond in a certain way
 * 5) Repeat
 * One chemical detection and one light detection needed

Person v. Bacteria
 * Most complex, based off of DNA paper (See here)
 * Applying ideas from DNA paper to bacteria
 * Create 8 strains of different antibiotic resistances
 * More feasible in E. coli than in Shewie

Lab

 * picked colonies
 * rehydrated MR-1 shewie strain

=Tuesday: June 17, 2008=
 * GFP/OD Measurement (results)
 * Make glycerol stocks
 * Miniprep vectors (protocol)
 * Make competent Shewie
 * Chemically competent (protocol) - actually used Zymo protocol which didn't work for Shewie and I don't think for E. coli
 * Electrocompetent (protocol) - actually used earlier protocol w/o sorbitol
 * Pour Cm plates
 * Transform Shewie with pACYC Duet and GFP vectors


 * align="center" style="background:#eeeeee;"|Name
 * align="center" style="background:#eeeeee;"|Registry Name
 * align="center" style="background:#eeeeee;"|Description
 * align="center" style="background:#eeeeee;"|Origin
 * align="center" style="background:#eeeeee;"|Size
 * align="center" style="background:#eeeeee;"|Marker


 * E1 || J23113||Low promoter GFP tester||pMB1||35||Kan
 * E2 || J23150||Medium promoter GFP tester||pMB1||35||Kan
 * E3 || J23151||High promoter GFP tester||pMB1||35||Kan
 * E4 || pACYC duet||Low-Medium copy vector||p15a||4008||Cm
 * }
 * For list of all parts, see here.
 * E4 || pACYC duet||Low-Medium copy vector||p15a||4008||Cm
 * }
 * For list of all parts, see here.
 * For list of all parts, see here.


 * Plasmid DNA Concentrations after Miniprep


 * align="center" style="background:#eeeeee;"|Name
 * align="center" style="background:#eeeeee;"|Registry Name
 * align="center" style="background:#eeeeee;"|Concentration (ng/uL)
 * E1a || J23113||67.1
 * E1b || J23113||76.2
 * E2a || J23150||45.4
 * E2b || J23150||72.4
 * E3a || J23151||41.8
 * E3b || J23151||68.5
 * }
 * For list of all parts, see here.
 * E3a || J23151||41.8
 * E3b || J23151||68.5
 * }
 * For list of all parts, see here.
 * }
 * For list of all parts, see here.

=Wednesday: June 18, 2008=

Protocols

 * Cloning Strategy
 * Punch out from registry


 * align="center" style="background:#eeeeee;"|Name
 * align="center" style="background:#eeeeee;"|Registry Name
 * align="center" style="background:#eeeeee;"|Description
 * align="center" style="background:#eeeeee;"|Origin
 * align="center" style="background:#eeeeee;"|Size
 * align="center" style="background:#eeeeee;"|Marker
 * E5 || pSB3K3||Low-Medium copy vector||p15a||2750||Kan
 * E6 || BBa-E1010||RFP only||pMB1||681||Kan
 * E7 || pSB1A2||GFP only||pMB1||2079||Amp
 * E8 || BBa_J04450||RFP with LacI promoter||pMB1||1069||Amp
 * E9 || BBa_J04430||GFP with LacI promoter||pMB1||1083||Amp
 * E10 ||BBa_I715038||T7 Polymerase with LacI promoter||pMB1||2878||Amp
 * }
 * For list of all parts, see here.
 * transform into E. coli TOP10 cells
 * E9 || BBa_J04430||GFP with LacI promoter||pMB1||1083||Amp
 * E10 ||BBa_I715038||T7 Polymerase with LacI promoter||pMB1||2878||Amp
 * }
 * For list of all parts, see here.
 * transform into E. coli TOP10 cells
 * transform into E. coli TOP10 cells


 * Make chemically and electrocompetent Shewie MR-1
 * Transform electrocompetent Shewie MR-1 and E. coli
 * P3, P4, P5, A1 in shewie
 * P3, P4, A1 in E. coli (TOP10)

Results

 * Results for Tuesday's (6/17) transformations
 * Results for Electrocompetent cells (first procedure no sorbitol)
 * P4 (pACYC) worked for both Shewie and E. coli; E. coli had a much higher density of colonies
 * None of the other plasmids worked for either Shewie or E. coli cells grew (likely due to low concentrations of cells)
 * Results for Chemically competent cells (w/ Zymo procedure)
 * No Shewie (5) or E. coli (2) cells grew

=Thursday: June 19, 2008=

Results from Electroporated Shewie and E. coli plates

 * Shewie
 * P3 J23151 (Kan) colonies grew and expressed GFP; colonies may have a pinkish tint; unexpected b/c origin of replication
 * P4 pACYC-Duet (Cm) colonies grew
 * P5 pSB3K3 (Kan) no growth, possibly due to the very small volume of DNA added
 * A1 (Cm) a few colonies grew
 * TOP10
 * P3 J23151 (Kan) - colonies grew
 * P4 pACYC-Duet (Cm) - a little growth
 * A1 (Cm) - colonies grew

Results TOP10 transformations w/ registry parts

 * P4 lawn
 * P5 no growth?
 * P6 no growth?
 * P7 many small colonies
 * P8 many small colonies
 * P9 many small colonies
 * P10 many small colonies

Restreaking electroporated E. coli plates
Some plates were overgrown (lawns/colonies too dense), so they were replated
 * P4 pACYC-Duet (Cm) restreaked from lawn onto 1000x dilution chloramphenicol plate)
 * P7 pSB1A2 one big and one small colony onto 2 ampicillin plates
 * P8 BBa_J04450 one big and one small colony onto 2 ampicillin plates
 * P9 BBa_J04430 one big and one small colony onto 2 ampicillin plates
 * P10 BBa_I715038 one big and one small colony onto 2 ampicillin plates

Some plates didn't have any visible colonies

S1 & E1 DNA Alignment
Thu Jun 19 15:45:18 2008 E. coli K12-DH108 from 1 to 1542 Alignment to Shewanella oneidensis MR-1 from 1 to 1529 Matches(|):1384 Mismatches(#):95 Gaps:113 Unattempted(.):0

Primers
1 aaattgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaa 60 |        ||||||||||||||||||||||||||||||||||||||||||||||||||            1 a-gtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaa 51 61 gtcgaacggta--ac-ag-gaagaagcttgct--tc-tttgctgacgagtggcggacggg 113 |||||#|||#| || || | ||    ||  |  || |##|#||#||||#||||||||||           52 gtcgagcggcagcacaagtg-agtt--tactcatgaggtggcgagcggcggacggg 104 114 tgagtaatgtct-gggaaactgcctga-tggagggggataacta-ctggaaacggta--g 168 |||||||||#|| ||| |#||||| #| |#|||||||||||| | #|||||||| |  |          105 tgagtaatgcctaggg-atctgcc-cagtcgagggggataac-agttggaaacg--actg 159 169 ctaataccgcataacgtcgc--a-agaccaaagagggggaccttcgggcctcttgccatc 225 |||||||||||| ||| | | | #|###||||||||||||#|||||||||||#||#||#          160 ctaataccgcat-acg-c-cctacgggggaaagagggggactttcgggcctctcgcgatt 216 226 ggatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatcc 285 |||||##||#||#||||||||||||||#||||#|||||#||||||||#||||||||||||         217 ggatgaacctaggtgggattagctagttggtgaggtaatggctcaccaaggcgacgatcc 276 286 ctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctac 345 |||||||#|||||||||||||#||||||||||||#||||||||||||#||||||||||||         277 ctagctgttctgagaggatgatcagccacactgggactgagacacggcccagactcctac 336 346 gggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgt 405 |||||||||||||||||||||||||||||||||#|#||#|||||||||||||||||||||         337 gggaggcagcagtggggaatattgcacaatgggggaaaccctgatgcagccatgccgcgt 396 406 gtatgaagaaggccttcgggttgtaaagtactttcagcggggagg-aagggagtaaagt- 463 ||#|||||||||||||||||||||||||#||||||||##|||||| |||| || ||||           397 gtgtgaagaaggccttcgggttgtaaagcactttcagtagggaggaaagg--gt-aagtc 453 464 -taatac--ctt-tgctcattgacgttacccgcagaagaagcaccggctaactccgtgcc 519 |||||| ||| | ||  #||||||||||##|||||||||#||||||||||||||||||          454 ctaatacgacttat-ct--gtgacgttacctacagaagaaggaccggctaactccgtgcc 510 520 agcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgca 579 ||||||||||||||||||||||||#|#|||||||||||||||||||||||||||||||##         511 agcagccgcggtaatacggagggtccgagcgttaatcggaattactgggcgtaaagcgtg 570 580 cgcaggcggtttgttaagtc-agatgtgaaatccccgggctcaacctgggaact-gcatc 637 |||||||||||||||||| | ||||||||||#|||#|||||||||||#|||| | ||||          571 cgcaggcggtttgttaag-cgagatgtgaaagccctgggctcaacctaggaa-tcgcat- 627 638 t--gatactg-gcaagcttgagtctcgtagaggggggtagaattccaggtgtagcggtga 694 | || |||| #||| ||#||||||#||||||||||||||||||||||||||||||||||          628 ttcga-actgaccaa-ctagagtcttgtagaggggggtagaattccaggtgtagcggtga 685 695 aatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacgaagactgac 754 ||||||||||||||||||||||||||||||||||||||||||||||||||#|||||||||         686 aatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgac 745 755 gctcaggtg--cgaaagcgtggggagcaaacaggattagataccctggtagtccacgccg 812 ||||| ||  |||||||||||||||||||||||||||||||||||||||||||||||||          746 gctca--tgcacgaaagcgtggggagcaaacaggattagataccctggtagtccacgccg 803 813 taaacgatgtcgacttggaggtt--gtgcccttgaggc-gt-ggct-tccggagctaacg 867 |||||||||||#|||#||| ||| ||| #||||| #| #| |||| ||  #||||||||          804 taaacgatgtctactcgga-gtttggtg-tcttga-acactgggctctc--a agctaacg 858 868 cgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacggg 927 |#||||||#|||||||||||||||||||||||||||||||||||||||||||||||||||         859 cattaagtagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacggg 918 928 ggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctg 987 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||#         919 ggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttaccta 978 988 gtcttgacatccacaga--actttccagagatg-g attggtgccttcgggaactgtgaga 1044 #|||||||||||||#|| ||  |#|||||||| |#|| ||||||||||||||#||||||          979 ctcttgacatccacggaagac--tgcagagatgcggtt-gtgccttcgggaaccgtgaga 1035 1045 caggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacg 1104 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||        1036 caggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacg 1095 1105 agcgcaacccttat-ctt-ttgttgccagc-ggt-ccggccgggaactcaaaggagactg 1160 ||||||||||#||| ||| | |||||||| #|| ##|| #||||||||#|#||||||||         1096 agcgcaacccctatccttat--ttgccagcacgtaat gg-tgggaactctagggagactg 1152 1161 ccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccag 1220 ||#|||||||||#|||||||||||||||#|||||||||||||||||||||||||||##||        1153 ccggtgataaaccggaggaaggtggggacgacgtcaagtcatcatggcccttacgagtag 1212 1221 ggctacacacgtgctacaatggcgca-tacaaagag---aagcgacctcgcgagag 1272 |||||||||||||||||||||||| | ||| ||||       ||||     |||||| |         1213 ggctacacacgtgctacaatggcg-agtac--agagggttgcaaagc-cgcgag-g 1263 1273 -caagcggacctcataaag -tgcgtcgtagtccggattggagtctgcaactcgactccat 1330 ##|||##|#||||#|||| | ||||||||||||||||||||||||||||||||||||||        1264 tggagctaatctcacaaagct-cgtcgtagtccggattggagtctgcaactcgactccat 1322 1331 gaagtcggaatcgctagtaatcgtggatcagaatgcca cggtgaatacgttcccgggcct 1390 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||        1323 gaagtcggaatcgctagtaatcgtggatcagaatgcca cggtgaatacgttcccgggcct 1382 1391 tgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaacct 1450 |||||||||||||||||||||||||||||||||#||||||||||||#|||||||||||||        1383 tgtacacaccgcccgtcacaccatgggagtgggctgcaaaagaagtgggtagcttaacct 1442 1451 tcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaacc 1510 |||||#|||||||#|||||||||||#|||||||||||||||||||||||||||||||#||        1443 tcgggggggcgctcaccactttgtggttcatgactggggtgaagtcgtaacaaggtagcc 1502 1511 gtaggggaacctgcggttggatcacctcctta 1542 #||||||||||||#||#||||||||||             1503 ctaggggaacctggggctggatcacct 1529

Transforming TOP 10 with the light sensor biobrick
We attempted to transform TOP 10 E. coli with the light sensor biobrick (to make P11). No colonies were visible the next morning. This is perhaps because the DNA turned pink after being punched out of the notebook.



=Friday: June 20, 2008=

Results from Thursday 6/19 Transformations

 * Shewie - no growth
 * E. coli
 * P3 lawn
 * P4 lawn
 * P5 no growth
 * P6 no growth

Transformed S1 via Electroporation
We used the electroporation protocol as noted in General Protocols with the following volumes of vector added:
 * P1 (J23113 low) - 2uL
 * P2 (J23150 med) - 2uL
 * P3 (J23151 high) - 2uL
 * P12 (pET Duet-1) - 5uL
 * P13 (pCDF Duet) - 1uL
 * P14 (pCOLA Duet) - 1uL

Put on the following plates at 30 degrees C over the weekend:
 * P1 (J23113 low) - Kan
 * P2 (J23150 med) - Kan
 * P3 (J23151 high) - Kan
 * P12 (pET Duet-1) - Amp
 * P13 (pCDF Duet) - Sm
 * P14 (pCOLA Duet) - Kan

Transformation of plasmids 11 and 15-20
Chemically competent E. coli DH5a were transformed with plasmids 15-20. Additionally, p11 was repeated.


 * We noticed that after punching out the holes from the notebook, the DNA usually turned purple. The DNA that remained yellow turned purple after adding EB buffer. The protocol in the notebook recommends heating the EB buffer before use, but we did not do this. Instead, we incubated the DNA and EB buffer for 20 minutes at 42 °C (instead of at room temperature).
 * Additionally, due to time constraints, we only incubated the DNA and bacteria in SOC medium for 1 hour.

Materials purchased
From McMaster-Carr

From FuelCellStore

Equipment Purchased
From Keithley