Team:Edinburgh/Notebook

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Edinburgh iGem 2008

 

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Contents

Calender

June
MTWTFSS
            [http://2008.igem.org/wiki/index.php?title=Edinburgh/1_June_2008&action=edit 1]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/2_June_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=Edinburgh/3_June_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=Edinburgh/4_June_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=Edinburgh/5_June_2008&action=edit 5] [http://2008.igem.org/wiki/index.php?title=Edinburgh/6_June_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=Edinburgh/7_June_2008&action=edit 7] [http://2008.igem.org/wiki/index.php?title=Edinburgh/8_June_2008&action=edit 8]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/9_June_2008&action=edit 9] [http://2008.igem.org/wiki/index.php?title=Edinburgh/10_June_2008&action=edit 10] [http://2008.igem.org/wiki/index.php?title=Edinburgh/11_June_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=Edinburgh/12_June_2008&action=edit 12] [http://2008.igem.org/wiki/index.php?title=Edinburgh/13_June_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=Edinburgh/14_June_2008&action=edit 14] [http://2008.igem.org/wiki/index.php?title=Edinburgh/15_June_2008&action=edit 15]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/16_June_2008&action=edit 16] [http://2008.igem.org/Edinburgh/17_June_2008 17] [http://2008.igem.org/wiki/index.php?title=Edinburgh/18_June_2008&action=edit 18] [http://2008.igem.org/Edinburgh/19_June_2008 19] [http://2008.igem.org/Edinburgh/20_June_2008 20] [http://2008.igem.org/wiki/index.php?title=Edinburgh/21_June_2008&action=edit 21] [http://2008.igem.org/wiki/index.php?title=Edinburgh/22_June_2008&action=edit 22]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/23_June_2008&action=edit 23] [http://2008.igem.org/Edinburgh/24_June_2008 24] [http://2008.igem.org/Edinburgh/25_June_2008 25] [http://2008.igem.org/Edinburgh/26_June_2008 26] [http://2008.igem.org/Edinburgh/27_June_2008 27] [http://2008.igem.org/Edinburgh/28_June_2008 28] [http://2008.igem.org/Edinburgh/29_June_2008 29]
[http://2008.igem.org/Edinburgh/30_June_2008 30]
July
MTWTFSS
  [http://2008.igem.org/Edinburgh/1_July_2008 1] [http://2008.igem.org/Edinburgh/2_July_2008 2] [http://2008.igem.org/Edinburgh/3_July_2008 3] [http://2008.igem.org/Edinburgh/4_July_2008 4] [http://2008.igem.org/Edinburgh/5_July_2008 5] [http://2008.igem.org/Edinburgh/6_July_2008 6]
[http://2008.igem.org/Edinburgh/7_July_2008 7] [http://2008.igem.org/Edinburgh/8_July_2008 8] [http://2008.igem.org/Edinburgh/9_July_2008 9] [http://2008.igem.org/Edinburgh/10_July_2008 10] [http://2008.igem.org/Edinburgh/11_July_2008 11] [http://2008.igem.org/Edinburgh/12_July_2008 12] [http://2008.igem.org/Edinburgh/13_July_2008 13]
[http://2008.igem.org/Edinburgh/14_July_2008 14] [http://2008.igem.org/Edinburgh/15_July_2008 15] [http://2008.igem.org/Edinburgh/16_July_2008 16] [http://2008.igem.org/Edinburgh/17_July_2008 17] [http://2008.igem.org/Edinburgh/18_July_2008 18] [http://2008.igem.org/Edinburgh/19_July_2008 19] [http://2008.igem.org/Edinburgh/20_July_2008 20]
[http://2008.igem.org/Edinburgh/21_July_2008 21] [http://2008.igem.org/Edinburgh/22_July_2008 22] [http://2008.igem.org/Edinburgh/23_July_2008 23] [http://2008.igem.org/Edinburgh/24_July_2008 24] [http://2008.igem.org/Edinburgh/25_July_2008 25] [http://2008.igem.org/Edinburgh/26_July_2008 26] [http://2008.igem.org/Edinburgh/27_July_2008 27]
[http://2008.igem.org/Edinburgh/28_July_2008 28] [http://2008.igem.org/Edinburgh/29_July_2008 29] [http://2008.igem.org/Edinburgh/30_July_2008 30] [http://2008.igem.org/Edinburgh/31_July_2008 31]
August
MTWTFSS
        [http://2008.igem.org/Edinburgh/1_August_2008 1] [http://2008.igem.org/wiki/index.php?title=Edinburgh/2_August_2008&action=edit 2] [http://2008.igem.org/Edinburgh/3_August_2008 3]
[http://2008.igem.org/Edinburgh/4_August_2008 4] [http://2008.igem.org/Edinburgh/5_August_2008 5] [http://2008.igem.org/Edinburgh/6_August_2008 6] [http://2008.igem.org/Edinburgh/7_August_2008 7] [http://2008.igem.org/Edinburgh/8_August_2008 8] [http://2008.igem.org/Edinburgh/9_August_2008 9] [http://2008.igem.org/Edinburgh/10_August_2008 10]
[http://2008.igem.org/Edinburgh/11_August_2008 11] [http://2008.igem.org/Edinburgh/12_August_2008 12] [http://2008.igem.org/Edinburgh/13_August_2008 13] [http://2008.igem.org/Edinburgh/14_August_2008 14] [http://2008.igem.org/Edinburgh/15_August_2008 15] [http://2008.igem.org/Edinburgh/16_August_2008 16] [http://2008.igem.org/Edinburgh/17_August_2008 17]
[http://2008.igem.org/Edinburgh/18_August_2008 18] [http://2008.igem.org/Edinburgh/19_August_2008 19] [http://2008.igem.org/Edinburgh/20_August_2008 20] [http://2008.igem.org/Edinburgh/21_August_2008 21] [http://2008.igem.org/Edinburgh/22_August_2008 22] [http://2008.igem.org/wiki/index.php?title=Edinburgh/23_August_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=Edinburgh/24_August_2008&action=edit 24]
[http://2008.igem.org/Edinburgh/25_August_2008 25] [http://2008.igem.org/Edinburgh/26_August_2008 26] [http://2008.igem.org/Edinburgh/27_August_2008 27] [http://2008.igem.org/Edinburgh/28_August_2008 28] [http://2008.igem.org/Edinburgh/29_August_2008 29] [http://2008.igem.org/wiki/index.php?title=Edinburgh/30_August_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=Edinburgh/31_August_2008&action=edit 31]

Summary of samples:

PCR products

P1: dxs from Eschaerichia coli JM109 (or K12?)
P2: appY from E. coli JM109 (or K12?)
P3: glgC from E. coli JM109 (or K12?)
P4: fusion PCR for BABEL1+glgC
P5: fusion PCR for BABEL2+glgC
P6: mutagenic PCR to remove EcoRI site 1 from glgC
P7: mutagenic PCR to remove EcoRI site 2 from glgC
P8: crtB (from Pantoea ananatis cells)
P9: crtI (from Douglas's maxiprep of the mutated crtIB plasmid)
P10: crtY (from P. ananatis cells). Failed.
P11: fusion PCR for rbs+dxs. Failed.
P12: repeat fusion PCR for rbs+dxs, using L11 and primers rbs2fclon1+pSB1A2insr1.
P13 and P14: MABEL mutation of site 1 on glgC mutant clones M19 and M22.
P15: fusion PCR for rbs+appY
P16, P17, P18 and P19: first attempts at cenA, cenB, cenC and cex.
P20, P21, P22 and P23: second attempts at cenA, cenB, cenC and cex.
P24, P25: repeat PCR for cenA and cex using Pfu. Failed.
P26: Positive control PCR with primers fd1 and rd1 to amplify 16S rRNA gene rrnB from Cellulomonas fimi.
P27: crtY from P. ananatis cells. - Failed
P28, P29: crtB and crtI from L18 and L19.
P30: Repeat positive control rrnB from C. fimi, annealing 1 minute.
P31: pZntA promoter
P32-P35: repeat PCR for cenA, cenB, cenC and cex, annealing 1 minute. Failed.
P36: M43 (glgC-mut1,2) with primers glgCf2/glgCr2 (25.07.08: AM)
P37: crtY from P. ananatis. (25.07.08: AM) - Failed
P38~P41: cenA, cenB, cenC and cex from heat-killed cell suspension.
P42~P45: cenA, cenB, cenC and cex from 'impure' DNA solution.
P46~P47: cenA and cex from 'impure' DNA solution, annealing 65C. Failed.
P48: crtY (new primer mixture) (previously P38a) (30.07.08: CF) - Failed
P49: Recreation of P12 (rbs+dxs) (previously P39a) (30.07.08: CF)
P50: third mutagenesis of glgc-mut1,2 (04.08.08: YAN)
P51: pCstA from E. coli cells (06.08.08: CF) - Failed
P52: cenA from heat killed C. fimi (07/08/08: YAN, AM) - Failed
P53: cex from heat killed C. fimi (07/08/08: YAN, AM) - Failed
P54: pCstA from E. coli cells - Purified (07.08.08: CF)

Ligations

L1: Edinbrick1+dxs
L2: Edinbrick1+appY
L3: Babel1+glgC
L4: Babel2+glgC
L5: Edinbrick1+appY repeat (initially labelled L4 in error)
L6: Self ligation of mutagenic PCR P6
L7: Self ligation of mutagenic PCR P7
L8: Ligation of synthetic ribosome binding site to M2 (BBa_K118000)
L9: Edinbrick1+crtB
L10: Edinbrick1+crtI (mutant lacking PstI sites, I hope)
L11: repeat ligation of synthetic ribosome binding site to M2 (BBa_K118000)
L12: rbs ligated with appY.
L13 and L14: self ligations of glgC mutation reactions P13 and P14.
L15: ligation of Edinbrick1 with rbs+dxs fusion PCR product (P12). - Failed
L16: Ligation of Edinbrick1 with rbs+appY fusion PCR product (P15) (17/07/08) - Failed
L17: Ligation of crtB (M36) to rbs (22/07/08)
L18: Ligation of crtI (M42) to rbs (22/07/08)
L19: pSB1A2+rbs+dxs ligation product (From P12) (25/07/08: Yan/OG)
L20: pSB1A2+rbs+appY ligation product (From P15) (25/07/08: Yan/OG)
L21: pSB1A2+rbs+crtB ligation product (From P28) (25/07/08: Yan/OG)
L22: pSB1A2+rbs+crtI ligation product (From P29) (25/07/08: Yan/OG)
L23: PzntA+pSB1A2 ligation product (From P31) (25/07/08: Yan/OG)
L24: Ligation of crtE to pSB1A2 (25/07/08: CF)
L25: Ligation of glgC double mutant (P36) to Edinbrick 1 (28.07.2008: AM)
L26: Ligation of Edi+P15 XP - rbs+appY (30.07.2008: HX)
L27: Ligation of Edi+P28 XP - rbs+crtB (30.07.2008: HX)
L28: Putative cenA ligated to Edinbrick1 (30.07.08: AM)
L29: Putative cex ligated to Edinbrick1 (30.07.08: AM)
L30: Self-ligation of P15 (3rd mutagenesis of glgC-mut1,2) (05.08.08: HX)
L31: Ligation of P36 (glgC-mut1,2, hopefully, could be P39) to Edinbrick1 (05.08.08: OG)
L32: Ligation of rbs+crtB (M67) to rbs+crtI (M50) to create crtB/crtI (07.08.08: AM, Yan)
L33: Ligation of PcstA (P54) to Edinbrick1 (08.08.08: OG)

Minipreps

M1 to M6: pSB1A2+dxs transformants. *M2, M3, M4 and M6* all looked correct on a gel. *M2* was sequenced to confirm identity and was maxiprepped as *X2*.
M7: Babel1+glgC transformant. Did not look right on gel.
M8 to M12: Babel2+glgC_ transformants. *M10 and M11* both looked good on a gel, but sequencing showed that both had the insert in the reverse orientation.
M13 to M18: pSB1A2+appY transformants. Gel 10 showed that M15 to M18, and possibly M14, all seemed to have inserts the right size.
M19 to M24: Babel2+glgC clones after mutation of EcoRI site 2. EcoRI digests on *Gel 11* suggest that *M19, M21 and M22* may have lost the EcoRI site.
M25 to M30: possible rbs+dxs transformants.
M31 to M36: crtB clones.
M37 to M42: crtI clones.
M43 to M47: possible glgC double mutants (both EcoRI sites gone)
M48: a possible rbs+dxs clone.
M49 to M54: pSB1A2-rbs+crtI
M55 to M60: PZntA promoter
M61 to M66: BABEL2+rbs+crtE
M67: rbs+crtB
M68 to M71: pSB1A2+glgC-mut1,2 (31.07.08: AM, YAN)
M72 to M75: pSB1A2+rbs+dxs (31.07.08: AM, YAN)
M76 to M79: BABEL2+crtE (31.07.08: AM, YAN)
M80 to M83: pSB1A2+rbs+appY (04/08/08: YAN and OG)
M84 to M85: pSB1A2+rbs+crtB (04/08/08: YAN and OG)
M86 to M90: pSB1A2+cenA (04/08/08: YAN and OG)
M91 to M96: pSB1A2+cex(04/08/08: YAN and OG)
M97 to M104: pSB1A2+cenA (05.08.08: YAN)
M105 to M108: pSB1A2+cex (05.08.08: YAN)

Maxipreps

X1: BioBrick J33201
X2: pSB1A2+dxs clone (as M2; BBa_K118000)
X3: pSB1A2+appY (from Plate 21, streak 3) (18 July 2008)
X4: pSB1A2+crtB (as M36; BBa_K118002) (25.07.08: A

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