IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week1/Chemical and Light

=Chemical=

6/23:



 * 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;"|Marker
 * align="center" style="background:#eeeeee;"|Observations
 * P1a || J23113||Low promoter GFP tester||pMB1||Kan||16 pink colonies of approx 7mm diameter
 * P2a || J23150||Medium promoter GFP tester||pMB1||Kan||no growth
 * P3a || J23151||High promoter GFP tester||pMB1||Kan||8 pink colonies of approx 7mm diameter
 * P12 || pETDuet-1||Low copy plasmid||pBR322-derived ColE1||Amp||non-specific Lawn; restreaks did not grow
 * P13 || pCDFDuet-1||Low copy plasmid||CloDF13||Sm||41 pink colonies and some small yellow ones that may be E. coli
 * P14 ||pCOLADuet-1||Low copy plasmid||ColA||Kan||No growth
 * }
 * P12 || pETDuet-1||Low copy plasmid||pBR322-derived ColE1||Amp||non-specific Lawn; restreaks did not grow
 * P13 || pCDFDuet-1||Low copy plasmid||CloDF13||Sm||41 pink colonies and some small yellow ones that may be E. coli
 * P14 ||pCOLADuet-1||Low copy plasmid||ColA||Kan||No growth
 * }
 * P14 ||pCOLADuet-1||Low copy plasmid||ColA||Kan||No growth
 * }

S1-P1A:

S1-P3A:

S1-P13:

6/23:
Picked colonies of E. coli and S1 and grew overnight at 37 and 30 degrees, respectively.




 * align="center" style="background:#eeeeee;"|Strain
 * align="center" style="background:#eeeeee;"|Plasmid Name
 * align="center" style="background:#eeeeee;"|Registry Name
 * align="center" style="background:#eeeeee;"|Plate Information
 * align="center" style="background:#eeeeee;"|Marker
 * align="center" style="background:#eeeeee;"|Colonies Picked
 * E. coli || P15||A1||6/18 electrocompetent||Cm||2
 * TOP10 || P4||pACYC duet||6/19 restreak electrocomp||Cm||2
 * TOP10 || P7||pSB1A2||6/19 restreak electrocomp big and small||Cm||2 (1 from big, 1 from small)
 * TOP10 || P8||BBa_J04450||6/19 restreak electrocomp big and small||Amp||2 (1 from big, 1 from small)
 * TOP10 || P9||BBa_J04430||6/19 restreak electrocomp big and small||Amp||2 (1 from big, 1 from small)
 * TOP10 || P10||BBa_I715038||6/19 restreak electrocomp big and small||Amp||2 (1 from big, 1 from small)
 * S1 || P4||pACYC duet||6/18 electrocomp||Cm||2
 * S1 || P21||A1||6/19 electrocomp||Cm||2
 * S1 || P13||pCDFDuet-1||6/20 electrocomp||Sm||2
 * S1 || P1a||J23113||6/20 electrocomp||Kan||3
 * S1 || P3a||J23151||6/20 electrocomp||Kan||3
 * }
 * S1 || P4||pACYC duet||6/18 electrocomp||Cm||2
 * S1 || P21||A1||6/19 electrocomp||Cm||2
 * S1 || P13||pCDFDuet-1||6/20 electrocomp||Sm||2
 * S1 || P1a||J23113||6/20 electrocomp||Kan||3
 * S1 || P3a||J23151||6/20 electrocomp||Kan||3
 * }
 * S1 || P1a||J23113||6/20 electrocomp||Kan||3
 * S1 || P3a||J23151||6/20 electrocomp||Kan||3
 * }
 * S1 || P3a||J23151||6/20 electrocomp||Kan||3
 * }

Total number of cultures: 24

6/24:
None of the samples in the LB amp media grew. All the others grew and then were miniprepped and stored in glycerol.


 * Glycerol stocks: 250 uL of culture in 500 uL of 20% LB glycerol.

Concentrations of DNA from minipreps:



 * align="center" style="background:#eeeeee;"|Plasmid
 * align="center" style="background:#eeeeee;"|Organism
 * align="center" style="background:#eeeeee;"|Concentration (ng/uL)
 * P1A a ||S1||92.54
 * P1A b ||S1||55.34
 * P1A c ||S1||58.21
 * P3A a ||S1||62.43
 * P3A b ||S1||168.41
 * P3A c ||S1||151.49
 * P4 ||S1||150.32
 * P13 ||S1||435.39
 * P21 ||S1||219.33
 * P4A a ||TOP10||49.81
 * P4A b ||TOP10||47.22
 * P21A a ||TOP10||61.16
 * P21A b ||TOP10||55.71
 * }
 * P13 ||S1||435.39
 * P21 ||S1||219.33
 * P4A a ||TOP10||49.81
 * P4A b ||TOP10||47.22
 * P21A a ||TOP10||61.16
 * P21A b ||TOP10||55.71
 * }
 * P4A b ||TOP10||47.22
 * P21A a ||TOP10||61.16
 * P21A b ||TOP10||55.71
 * }
 * P21A b ||TOP10||55.71
 * }

6/26: Amp cultures still did not grow in liquid media, neither at 1:1000 or a 1:2000 dilution of amp

6/24:
P5 and P6 were punched from the book and transformed in E3 (the death resistant strain)

6/25: No Growth

6/24:
Performed PCR as per protocol listed in General Protocols.

Primer Sets
 * 1 = Shewanella F and Shewanella R
 * 2 = E. coli F and E. coli R
 * 3 = Common F and Common R




 * align="center" style="background:#eeeeee;"|Tube #
 * align="center" style="background:#eeeeee;"|Strain/ Colony of Template DNA
 * align="center" style="background:#eeeeee;"|Primer Set Used
 * align="center" style="background:#eeeeee;"|Volume of Template DNA
 * align="center" style="background:#eeeeee;"|Volume of Primer
 * align="center" style="background:#eeeeee;"|Volume of Supermix
 * align="center" style="background:#eeeeee;"|Volume of Water
 * 1|| S1 P1a||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 2|| S1 P1a||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 3|| S1 P1a||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 4|| S1 P1a||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 5|| S1 P1a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 6|| S1 P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * 7|| S1 P3a||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 8|| S1 P3a||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 9|| S1 P3a||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 10|| S1 P3a||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 11|| S1 P3a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 12|| S1 P3a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * 13|| S1 MR-1||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 14|| S1 MR-1||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 15|| S1 MR-1||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 16|| S1 MR-1||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 17|| S1 MR-1||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 18|| S1 MR-1||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * 19|| E. coli P1a||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 20|| E. coli P1a||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 21|| E. coli P1a||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 22|| E. coli P1a||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 23|| E. coli P1a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 24|| E. coli P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * }
 * 13|| S1 MR-1||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 14|| S1 MR-1||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 15|| S1 MR-1||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 16|| S1 MR-1||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 17|| S1 MR-1||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 18|| S1 MR-1||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * 19|| E. coli P1a||1 (SF & SR)||1uL||1uL (each)||45uL||2uL
 * 20|| E. coli P1a||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 21|| E. coli P1a||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 22|| E. coli P1a||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 23|| E. coli P1a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 24|| E. coli P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * }
 * 20|| E. coli P1a||1 (SF & SR)||3uL||1uL (each)||45uL||2uL
 * 21|| E. coli P1a||2 (EF & ER)||1uL||1uL (each)||45uL||2uL
 * 22|| E. coli P1a||2 (EF & ER)||3uL||1uL (each)||45uL||2uL
 * 23|| E. coli P1a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 24|| E. coli P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * }
 * 23|| E. coli P1a||3 (CF & CR)||1uL||1uL (each)||45uL||2uL
 * 24|| E. coli P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * }
 * 24|| E. coli P1a||3 (CF & CR)||3uL||1uL (each)||45uL||2uL
 * }

6/25
Gel results from PCR:



Key:



Note: S1 P3a With Primer Set 2 and 3 uL of template DNA evaporated during PCR so was not included in the gel.
 * align="center" style="background:#eeeeee;"|Well #
 * align="center" style="background:#eeeeee;"|Strain/ Colony
 * align="center" style="background:#eeeeee;"|Primer Set Used
 * align="center" style="background:#eeeeee;"|Volume of Template DNA
 * 1|| 1 Kb ladder||N/A||N/A
 * 2|| S1 P3a||1 (SF & SR)||3uL
 * 3|| S1 P3a||1 (SF & SR)||1uL
 * 4|| S1 P3a||2* (EF & ER)||1uL
 * 5|| S1 P3a||3 (CF & CR)||1uL
 * 6|| S1 P3a||3 (CF & CR)||3uL
 * 7|| S1 P1a||1 (SF & SR)||3uL
 * 8|| S1 P1a||1 (SF & SR)||1uL
 * 9|| S1 P1a||2 (EF & ER)||1uL
 * 10|| S1 P1a||2 (EF & ER)||3uL
 * 11|| S1 P1a||3 (CF & CR)||3uL
 * 12|| S1 P1a||3 (CF & CR)||1uL
 * }
 * 7|| S1 P1a||1 (SF & SR)||3uL
 * 8|| S1 P1a||1 (SF & SR)||1uL
 * 9|| S1 P1a||2 (EF & ER)||1uL
 * 10|| S1 P1a||2 (EF & ER)||3uL
 * 11|| S1 P1a||3 (CF & CR)||3uL
 * 12|| S1 P1a||3 (CF & CR)||1uL
 * }
 * 11|| S1 P1a||3 (CF & CR)||3uL
 * 12|| S1 P1a||3 (CF & CR)||1uL
 * }
 * 12|| S1 P1a||3 (CF & CR)||1uL
 * }



Key:



Note: There was a problem with the gel in Well 10, which most likely accounts for the lighter band.
 * align="center" style="background:#eeeeee;"|Well #
 * align="center" style="background:#eeeeee;"|Strain/ Colony
 * align="center" style="background:#eeeeee;"|Primer Set Used
 * align="center" style="background:#eeeeee;"|Volume of Template DNA
 * 1|| 1 Kb ladder||N/A||N/A
 * 2|| S1 MR-1||1 (SF & SR)||1uL
 * 3|| S1 MR-1||2 (EF & ER)||1uL
 * 4|| S1 MR-1||3 (CF & CR)||1uL
 * 5|| 100 bp Ladder||N/A||N/A
 * 6|| E.coli P1a||1 (SF & SR)||1uL
 * 7|| E.coli P1a||1 (SF & SR)||3uL
 * 8|| E.coli P1a||2 (EF & ER)||1uL
 * 9|| E.coli P1a||2 (EF & ER)||3uL
 * 10*|| E.coli P1a||3 (CF & CR)||1uL
 * 11|| E.coli P1a||3 (CF & CR)||3uL
 * 12|| 1 Kb Ladder||N/A||N/A
 * }
 * 7|| E.coli P1a||1 (SF & SR)||3uL
 * 8|| E.coli P1a||2 (EF & ER)||1uL
 * 9|| E.coli P1a||2 (EF & ER)||3uL
 * 10*|| E.coli P1a||3 (CF & CR)||1uL
 * 11|| E.coli P1a||3 (CF & CR)||3uL
 * 12|| 1 Kb Ladder||N/A||N/A
 * }
 * 11|| E.coli P1a||3 (CF & CR)||3uL
 * 12|| 1 Kb Ladder||N/A||N/A
 * }
 * 12|| 1 Kb Ladder||N/A||N/A
 * }

Remy's pZ vector system Plasmids
[[Media:Bujard lutz.pdf | Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements]]

6/25: transfer colonies to liquid media

6/26: mini prep plasmids




 * 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;"|Marker
 * align="center" style="background:#eeeeee;"|Regulatory unit
 * align="center" style="background:#eeeeee;"|Concentration ng/uL
 * P27 ||pZS4int2||repressor plasmid for chromosomal integration||pSC101||Sm||Constitutive expression of LacI and TetR||
 * P28 ||pZE21||GFP||ColE1||Kan||PLtctO-1||
 * P29 ||pZS*2R||Venus (mutant YFP)||pSC101*||Kan||PL (Plambda)||
 * P30 ||pZA32||YFP||p15A||Cm||PLlacO-1||
 * P31 ||pZS*1R||Venus||pSC101*||amp||PL (Plambda)||
 * P32 ||pZA32||Venus||p15A||Cm||PLlacO-1||
 * P33 ||pZA31||Venus||p15A||Cm||PLtctO-1
 * P34 ||pZE12||GFP||ColE1||Amp||PLlacO-1||
 * P35 ||pLDR8||Accessory for chromosomal integration||??||???||??||
 * }
 * P32 ||pZA32||Venus||p15A||Cm||PLlacO-1||
 * P33 ||pZA31||Venus||p15A||Cm||PLtctO-1
 * P34 ||pZE12||GFP||ColE1||Amp||PLlacO-1||
 * P35 ||pLDR8||Accessory for chromosomal integration||??||???||??||
 * }
 * P34 ||pZE12||GFP||ColE1||Amp||PLlacO-1||
 * P35 ||pLDR8||Accessory for chromosomal integration||??||???||??||
 * }
 * }

Also two strains with integrated copies of repressors, low level constitutive promoters


 * MC4100 Z1 - repressor lac and tet (Sm resistant)
 * DH10B Z2 - repressor lac and tet (Sm resistant)

6/26:
Re-transformed BioBrick parts in E1 because amp plates weren't working. Also re-transformed P5 and P6 in E3.




 * 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;"|Marker
 * align="center" style="background:#eeeeee;"|Transformed? (6/26)
 * align="center" style="background:#eeeeee;"|Picked colonies?
 * align="center" style="background:#eeeeee;"|Miniprepped?
 * align="center" style="background:#eeeeee;"|Made glycerol stocks?
 * P7 || pSB1A3*||High copy plasmid with Amp resistance||ColE1||Amp||Failed in E1|| || ||
 * P8 || BBa_J04450||RFP with LacI promoter||pMB1||Amp||Failed in E1|| || ||
 * P9 || BBa_J04430||GFP with LacI promoter||pMB1||Amp||Failed in E1|| || ||
 * P10 || BBa_I715038||T7 Polymerase with LacI promoter||pMB1||Kan/Amp||Failed in E1|| || ||
 * P36 || BBa_I763004||GFP + IPTG + LVA||||Kan/Amp||Failed in E1|| || ||
 * P5 (2007) ||pSB3K3*||Low-Medium copy vector (w/ death gene)||p15A||Kan||Yes, in E3||2 (A and B spilled)|| ||
 * P6 (2007) ||BBa-E1010||RFP only (w/ death gene)||pMB1||Kan||Failed in E3|| || ||
 * P5 (2008) ||pSB3K3*||Low-Medium copy vector (w/ death gene)||p15A||Kan||Failed in E3|| || ||
 * }
 * P36 || BBa_I763004||GFP + IPTG + LVA||||Kan/Amp||Failed in E1|| || ||
 * P5 (2007) ||pSB3K3*||Low-Medium copy vector (w/ death gene)||p15A||Kan||Yes, in E3||2 (A and B spilled)|| ||
 * P6 (2007) ||BBa-E1010||RFP only (w/ death gene)||pMB1||Kan||Failed in E3|| || ||
 * P5 (2008) ||pSB3K3*||Low-Medium copy vector (w/ death gene)||p15A||Kan||Failed in E3|| || ||
 * }
 * P5 (2008) ||pSB3K3*||Low-Medium copy vector (w/ death gene)||p15A||Kan||Failed in E3|| || ||
 * }
 * }

Notes:
 * Control was also done with untransformed E3 on LB plates.
 * pSB1A3 was used instead of pSB1A2 because the Parts Registry lists pSB1A3 as the replacement for pSB1A2.
 * P5 and P6 were taken from Spring 2007 plates, and iGEM 2008 P5 was punched out and transformed again.

6/27:
P5 (2007) plate was put in incubator for longer, and will be picked on Sunday along with the other transformations done today.




 * 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;"|Marker
 * align="center" style="background:#eeeeee;"|Transformed? (6/27)
 * align="center" style="background:#eeeeee;"|Picked colonies? (6/29)
 * align="center" style="background:#eeeeee;"|Miniprepped?
 * align="center" style="background:#eeeeee;"|Made glycerol stocks?
 * P12 || pETDuet-1||Low copy plasmid||pBR322-derived ColE1||Amp||in E1||2|| ||
 * P37 ||BBa_I722007||Constructive expression LacI w/ pTetR promoter||||Amp||in E1|| 2 (A and B didn't work)|| ||
 * P38 ||BBa_J23114||High constitutive promoter||||Amp||in E1|| 2|| ||
 * P38 (2007) ||BBa_J23114||High constitutive promoter||||Amp||in E1|| 2 (A and B didn't work)|| ||
 * P39 ||BBa_J23113||Low constitutive promoter||||Amp||in E1||2 (B didn't work) || ||
 * P39 (2007) ||BBa_J23113||Low constitutive promoter||||Amp||in E1|| 2 (B did not work)|| ||
 * P40 ||BBa_B0032||Ribosome Binding Site||||Amp||in E1||2 (B didn't work) || ||
 * P40 (2007) ||BBa_B0032||Ribosome Binding Site||||Amp||in E1|| 2 (A did not work)|| ||
 * P41 ||BBa_B0010||Transcriptional Terminator||||Amp||in E1||2 (A and B didn't work) || ||
 * P41 (2007) ||BBa_B0010||Transcriptional Terminator||||Amp||in E1|| 2 (Neither worked)|| ||
 * P42 ||BBa_C0012||LacI coding region||||Amp||in E1||2 (A and B didn't work) || ||
 * P42 (2007) ||BBa_C0012||LacI coding region||||Amp||in E1|| 1 (didn't work)|| ||
 * P43 ||||TetR coding region||||Amp||in E1||2 (A and B didn't work) || ||
 * P43 (2007) ||||TetR coding region||||Amp||in E1|| 2 (B didn't work)|| ||
 * P44 ||BBa_C0051||cI lambda||||Amp||in E1|| 2 (A and B didn't work)|| ||
 * P44 (2007) ||BBa_C0051||cI lambda||||Amp||in E1|| 2 (B didn't work)|| ||
 * P45 ||BBa_E0240||GFP only w/ RBS & terminator||||Amp||in E1||2 (A and B didn't work)|| ||
 * P45 (2007) ||BBa_E0240||GFP only w/ RBS & terminator||||Amp||in E1|| 2|| ||
 * P46 ||BBa_I51020||Base Vector||||Amp||in E3||2 || ||
 * P47 ||BBa_P1003||Kan Resistance Cassette||||Kan||No in E1|| 0|| ||
 * P47 (2007) ||BBa_P1003||Kan Resistance Cassette||||Kan||in E1|| 2 (A and B spilled)|| ||
 * P48 ||BBa_P1004||Cm Resistance Cassette||||Cm||No in E1|| 0|| ||
 * P48 (2007) ||BBa_P1004||Cm Resistance Cassette||||Cm||in E1||2 || ||
 * P49 ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2 (B didn't work) || ||
 * P49 (2007) ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2|| ||
 * P50 ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2 (A and B didn't work) || ||
 * P50 (2007) ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2|| ||
 * }
 * P44 ||BBa_C0051||cI lambda||||Amp||in E1|| 2 (A and B didn't work)|| ||
 * P44 (2007) ||BBa_C0051||cI lambda||||Amp||in E1|| 2 (B didn't work)|| ||
 * P45 ||BBa_E0240||GFP only w/ RBS & terminator||||Amp||in E1||2 (A and B didn't work)|| ||
 * P45 (2007) ||BBa_E0240||GFP only w/ RBS & terminator||||Amp||in E1|| 2|| ||
 * P46 ||BBa_I51020||Base Vector||||Amp||in E3||2 || ||
 * P47 ||BBa_P1003||Kan Resistance Cassette||||Kan||No in E1|| 0|| ||
 * P47 (2007) ||BBa_P1003||Kan Resistance Cassette||||Kan||in E1|| 2 (A and B spilled)|| ||
 * P48 ||BBa_P1004||Cm Resistance Cassette||||Cm||No in E1|| 0|| ||
 * P48 (2007) ||BBa_P1004||Cm Resistance Cassette||||Cm||in E1||2 || ||
 * P49 ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2 (B didn't work) || ||
 * P49 (2007) ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2|| ||
 * P50 ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2 (A and B didn't work) || ||
 * P50 (2007) ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2|| ||
 * }
 * P48 ||BBa_P1004||Cm Resistance Cassette||||Cm||No in E1|| 0|| ||
 * P48 (2007) ||BBa_P1004||Cm Resistance Cassette||||Cm||in E1||2 || ||
 * P49 ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2 (B didn't work) || ||
 * P49 (2007) ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2|| ||
 * P50 ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2 (A and B didn't work) || ||
 * P50 (2007) ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2|| ||
 * }
 * P49 (2007) ||BBa_P1002||Amp Resistance Cassette||||Amp||in E1|| 2|| ||
 * P50 ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2 (A and B didn't work) || ||
 * P50 (2007) ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2|| ||
 * }
 * P50 (2007) ||BBa_P1005||Tet Resistance Cassette||||Amp||in E1|| 2|| ||
 * }

6/24
Plates streaked and put in incubator.

6/25:
Top Left: E1 Bottom Left: E1-P1a Top Right: S1 Bottom Right: S1-P1a

Top Left: chemically competent E. coli Bottom Left: E1-P3 Top Right: WT S1 Bottom Right: S1-P3

Top Left: E1 Bottom Left: E1-A1 (P21) Top Right: S1 Bottom Right: S1-A1 (P21)

6/25:
Added lactate and birnesite to LB in a vial, bubbled N2 gas to degas it and then added Shewie at concentrations of 100 uL and 500 uL, while keeping a control with no Shewie added. Vials were placed in 30 degree shaking incubator overnight.

6/26:
All vials, including the control, turned light yellow (the color of the LB). This was most likely due to contamination, which was partly confirmed by looking at the vials through the microscope. This was not unexpected because the vials we used had not been autoclaved, and it was very difficult to maintain sterile conditions and precise concentrations of birnesite/ lactate.

Questions/ Concerns
Regarding transformations of BioBrick Parts:
 * 1) Given the parts that we've succeeded in transforming in either E. coli, or Shewanella, or both, what is our plan for designing vectors? How many plasmids will we need?
 * 2) How should we incorporate Remy's plasmids into those designs?
 * 3) To what extent can we take parts from or use the Duet vectors?
 * 4) Can we confirm that pMB1 does work in Shewanella, contrary to the 1997 paper that suggests otherwise?
 * 5) If we have successful transformations with both the 2007 and 2008 versions of a BioBrick part, should we prefer one over the other?

Regarding anaerobic Shewie growth: (Many of these questions may be the same as those posed by the Widgetry group.)
 * 1) What are the optimal concentrations of lactate and birnesite that we should use?
 * 2) Tips on maintaining sterility/ preventing contamination?
 * 3) Orianna's paper lists three different media that she used for various purposes-- what does our basic media need?
 * 4) How much Shewie should we be using? Should we be adding them at a certain growth phase or OD?

=Light=

Transformations of P11, P15-20
ENDED IN FAILURE: SEE NEW TRANSFORMATION BELOW

Colony counts
6/23:

Restreaking
06/23: P15, which has GFP under a constitutive Tet promoter did not appear to fluoresce (none of the fluorescent constructs did), so we restreaked all the of the plates (with the same antibiotic on which they were originally grown). They were left in the 37 degree incubator along with a blank Kan and a blank Amp plate (- controls).

06/24: All of the restreaked plates grew and were streaked to individual colonies. Both the Kan and Amp negative controls (no cells) were blank. However, p15 (GFP under pTet) did not appear to glow.

Overnight cultures
06/23: The above was repeated in 5mL LB liquid cultures instead of plates.


 * 06/24: None of the cultures in LB Amp grew (E1P17 in LB Kan did grow). We recultured the other samples in LB Amp and made a master plate with patches from the same colonies that were cultured. Since others seemed to get similar problems (no growth in liquid culture but growth on plates for Amp), we made fresh LB Amp (from freshly made Amp stock) and repeated the cultures.


 * 06/25: These LB-Amp cultures did not grow in the new medium either. We plated them on new LB amp plates (new plates 1X amp or 1/1000) and cultured them in low concentration LB amp (0.5X or 1/2000 amp).


 * 06/26: None of the cultures grew on either of the liquid media (1X and .5X LB-Amp). Additionally, none of the master plate colonies restreaked onto the new LB-Amp plates that we poured grew. This suggests to us that the original LB-Amp plates were not selective enough (perhaps Amp broke down). As a control that our bacteria were capable of growth, we inoculated a plain LB liquid culture with each of the 6 colonies from the master plate. This culture did grow (cloudy). A plain LB liquid culture left as a negative control did not.

Miniprep of P17
ENDED IN FAILURE: SEE NEW TRANSFORMATION BELOW

06/24: Miniprep of E1P17. The DNA is in the -20 °C iGEM freezer. The DNA concentration is 54.8 ng/μL.

Restriction Digest
06/24: Digest of P17 DNA with Xba1 and Spe1. The entire plasmid is 4425 bp and the short fragment (containing only the BioBrick) is 902 bp.

Digest mixture (incubated overnight at 37 °C):

15 μl DNA

6.25 μl water

2.5 μl NEB Buffer 2

0.25 μl 100X BSA

1 μl Xba1

1 μl Spe1

Gel of digest products (6/25)

06/25: Since the previous digest was unsuccessful, we digested the same plasmid with different combinations of restriction enzymes. We also tested two other plasmids from the chemical group (P4 and P21). The digest mixtures were the same as above (6/24) with the appropriate enzyme swapped in. For the single digests, the volume of water was 6.75 μl and all else remained constant. Again, incubation is overnight at 37 °C.

From the lanes with P17, which again indicate that the plasmid we have from the miniprep is not the right size, we conclude that the P17 transformation failed. It is being repeated (see below).

Transformations of P22-24
ENDED IN FAILURE: SEE NEW TRANSFORMATION BELOW

06/24: Transformations occurred in duplicate (TOP10 and DH5α) with Amp selection.

06/25: All plates had colonies. We inoculated liquid cultures of cells with each of these plasmids in low (0.5X or 1/2000 amp) and high (1X or 1/1000 amp) concentration LB amp.

E1P22: 376 colonies E1P23: 352 colonies E1P24: 392 colonies E2P22, E2P23, and E2P24 had too many colonies to count.


 * 06/26: None of the liquid cultures (both high and low Amp) had any growth. Additionally, we patched the colonies we were growing on a master plate that was of the new LB-Amp plates that we poured. No colonies grew on this plate either. This again seems to indicate that the initial transformation plates did not have enough selection (perhaps Amp was degraded).

Transformations of P17, 20, 25, 26
SEE NEW TRANSFORMATION BELOW

06/25: Transformed P17, P20, P25, and P26 in DH5α cells. P17 and P20 have Amp and Kan resistance, but since AmpR cells don't seem to grow in the liquid media, we're repeating the process by plating the cells on Kan (and eventually grow them on LB-Kan liquid medium). P25 and P26 are new and have Amp resistance.

06/26: P20 (kan plate) had 4 colonies and P25 (amp plate) had 1 colony. The other plates (with P17, P20, and P26) did not have any colonies. The kan and amp negative control plates (DH5α cells with no DNA) also had no colonies.

New transformations: P11, P15-20, P22-26
06/26

We transformed TOP10 cells using parts from both the 2008 and 2007 registries, apart from P11 and P19 which were not available in the 2007 registry.

The protocol for getting DNA from the 2007 registry is as follows:

1. Puncture a hole in the foil with a pipet.

2. Add 15 μl H2O and wait for about 5 minutes.

3. Add 1 μl DNA to 50 μl bacteria.

P17 and P20 were plated on Kan plates. All other samples were on Amp plates that we made (at 1X or 1/1000 amp).

Transformation results
Both negative plate controls (Amp and Kan) did not have any colonies. The "mock transformations" (no DNA) were also negative. The positive plate control had a sample of pEZ12 GFP from Remy and this plasmid also carried Amp resistance. There was a lawn of bacteria on this plate. We also did a positive transformation control that had 1 μg pUC19 DNA for 50 μl TOP10 cells. This DNA came with the TOP10 cells from Invitrogen and there were 26 colonies on the plate.

6/10 of the 2007 parts appear to have transformed (colonies grew) and 1/12 of the 2008 parts appear to have transformed successfully.

Remy's positive control with GFP was fluorescent, along with P15 (GFP with Tet promoter) and P20 (LVA-tagged GFP with Lac promoter). This indicates that the transformations of P15 and P20 were successful.

Set up liquid cultures (with master plates) for all of the plasmids that had colonies.

Minipreps and digests
6/28: The overnight cultures grew successfully and were mini-prepped. Note that there are replicates (e.g. 15A and 15B are different tubes from the overnight cultures. * and *-less denotes replicates during minipreppring). Unfortunately, I forgot to keep culture for glycerol stocks. I performed a NotI digest (see recipe under "RE Digest")- it's the standard single digest recipe, but halfed. The digest ran for the evening, and the results are below:

All of the P15 samples appear to have worked properly. P16 is questionable.

RE digest
06/27 These digests were meant to test the enzymes and digest conditions. P3 and P1 were supposed to be controls. However they were Biobricks miniprepped from Shewie and it doesn't look like the transformation into Shewie worked as even the uncut DNA is not the right size. We will redo the digest with DNA from E. coli.

I set up another overnight digest but doubled the overall volume so that there was 1 μl of the restriction enzyme (NotI).

30 μl DNA

13.5 μl H2O

5 μl 10X NEB buffer 3

0.5 μl 100X BSA

1 μl NotI

The bands in the cut P1-3 appear to be of the expected size. However, running a sample of the uncut miniprep gives more than one band (is this b/c an unclean miniprep?) P17 continues to digest as if it is a biobrick. However, the bands continue to be of unexpected sizes.