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
Ligation products
Minipreps
Maxipreps
Plates
Gels

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

h5. Gels

  • Gel 1:* PCR reactions P1, P2, P3
  • Gel 2:* minipreps M1 to M6,Plate 119/120: Transformation of L40 (CenA+Edinbrick1) (14.08.08: YAN)
  • Gel 3:* minipreps M2, M3, M4, M6, EcoRI, plus L1 and L2
  • Gel 4:* minipreps M7 to M12, undigested
  • Gel 5:* minipreps M7 to M12, EcoRI/PstI
  • Gel 6:* minipreps M2, M3, M4, M6, HindIII; also P4 and P5 (fusion PCR)
  • Gel 7:* minipreps M10 and M11, EcoRI (two versions of this gel)
  • Gel 8:* PCR products P6 and P7 (mutagenic PCR on M11)
  • Gel 9:* maxipreps X1 and X2, etc.
  • Gel 10:* minipreps M13 to M18, E/P digests.
  • Gel 11:* minipreps M19 to M24, EcoRI digests, plus PCR P11.
  • Gel 12:* minipreps M25 to M30 of possible rbs+dxs clones: none look right.
  • Gel 13:* PCR P13 and P14 to mutated second EcoRI site on glgC clones M19 and M22.
  • Gel 14:* minipreps M31 to M42 (crtB and crtI clones) E/P, and P15 (rbs+appY)
  • Gel 15:* redigest of M32, M36 (crtB), M39, M40 (crtI) E/P; unsuccessful.
  • Gel 16:* purified PCR products for cenA, cenB, cenC, cex (P16, 17, 18, 19); unsuccessful.
  • Gel 17:* PCR products for cenA, cenB, cenC, cex (P20, 21, 22, 23) ~ second attempt; unsuccessful.
  • Gel 21:* P26 (rrnB from C fimi); unsuccessful.
  • Gel 22:* P27 (crtY ~ unsuccessful), P28, P29 (crtB, crtI ~ successful).
  • Gel 23:* P30 (repeat rrnB from C fimi) and PZNTA. Successful.
  • Gel 25:* P36 (glgC excision ~ successful), P37 (crtY ~ failed).
  • Gel 26:* P38~43 (cenA, cenB, cenC, cex from heat-killed cell suspension; cenA and cenB from 'impure' DNA solution.
  • Gel 27:* Repeat of Gel 26 but stained with SYBRsafe; putative cenA and cex fragments cut out.
  • Gel 28:* M49~54 (pSB1A2-rbs+crtI) digested with EcoRI.
  • Gel 29:* M55~60 (PZntA), M61~66 (CrtE+RBS) all digested with EcoRI and PstI.
  • Gel 30:* P46 (cenA), P47 (cex) from heat killed cell soln, annealing 65C. Failed. (30/07/08)
  • Gel 31*: M49 (X,SX,sac/spt), M50(X,SX,sac/spt), M63(E,EP), M67(E/P, sac1/spe1), P38(_cenA_). (30/07/08: OG)
  • Gel 32:* M55 to M60 double digestion with EcorI and PstI, and single digestion with EcoRI only. (30/07/08: Yan)
  • Gel 33:* M68~71 (pSB1A2-glgC m1+m2) digested with EcoRI+PstI, M72~75 (pSB1A2-rbs+dxs) digested with EcoRI, M76~79 (Babel2\+_crtE_) digested with EcoRI+PstI. See 01.08.2008 entry for results.
  • Gel 34:* M68~69 (pSB1A2-glgC m1+m2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2-rbs+dxs) digested with EcoRI alone and SacI+SpeI. See 01.08.2008 entry for results.
  • Gel 35:* P36 and L25. P36 successful, L25 yields clear glgC-length band but faint vector-length band.
  • Gel 36:* Double digestion of M80 to M96 with EcoRI and PstI, and PCR production of third mutagenesis of glgc mutant 1&2. Results were good for M80-82 (rbs\-_appY_\-pSB1A2), M84-85 (rbs\-_crtB_\-pSB1A2), M92 (_cex_\-pSB1A2) and P50 (_glgC_\-mut1,2+3). Other results were poor. (YAN&OG).
  • Gel 37:* Double digestion of M97 to M108 with EcoRI and PstI.
  • Gel 39:* Analytical digests using a) EcoRI and b) EcoRI/PstI of M70, M71 (both pSB1A2\-_glgC_ mut1+2) and M92 (pSB1A2\-_cex_). Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (06/08/08: CF)
  • Gel 40* PCR products of PcstA (P54) (and SOB). Results look promising (fairly prominent band <500bp). (07.08.08: CF)
  • Gel 41:* PCR products of P55 to P58 (11.08.08: YAN)
  • Gel 42:* _C. fimi_ genomic DNA and PCR products P61~P63 from _C. fimi_ genomic DNA (11.08.08: CF)
  • Gel 43:* Double digests of BABEL2\-_crtE_ (M79) and Edinbrick1 with EcoRI and PstI, showing a single band at \~3kb for BABEL2\+_crtE_ and bands at \~2.5kb and 800bp for Edinbrick1. (11.08.08: AH)
  • Gel 44:* Double digests of BABEL1-glgc mut 1+2+3 (M117 to M120), pSB1A2-glgc mut 1+2 (M113-M116) and pSB1A2-CrtBI with EcoRI and PstI.(11.08.08: YAN)
  • Gel 45:* P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (zm1/ISA1) and P67 (zm2/ISA2). (12.08.08: CF/AM)
  • Gel 46:* Double digests of M121 to M126: M121 to M123 (pSB1A2-CrtY-1 to pSB1A2-CrtY-3), M124 to M126 (pSB1A2-CrtY-RBS-1 to pSB1A2-CrtY-RBS-3) with EcoRI and PstI. (12.08.08: YAN)
  • Gel 47:* P57 and P64 (cex) PstI digests and P65 (cenA) (12.08.08: AM)
  • Gel 48:* M109 and M110 (_crtBI_) BamHI/EcoRV digest, M115 and M116 (pSB1A2\+_glgC_\-mut1,2) HindIII digest M115 and M116 (pSB1A2\+_glgC_\-mut1,2) EcoRI/HindIII digest. (13.08.08: AH)
  • Gel 49:* P68 (cenB) and P69 (cenC) from _C. fimi_ genomic DNA. Failed. (13.08.08: AM)
  • Gel 50:* M127~M138? Please confirm. (13.08.2008: OG, I believe, OG: i think this is the one when me andy and yan were loading it and taking pictures for the webpage.)
  • Gel 51:* Undocumented gel stained with SYBR-safe with bands for purification?
  • Gel 52:* P70 (glgC mut1+2), P71 (glgC mut 1+2+3). (14.08.2008: AM)
  • Gel 53:* Minipreps of M139 to M143 (CrtE+PSB1A2) (15.08.08: YAN/OG)
  • Gel 54:* P71A (glgC mut 1+2), P72 (glgC mut 1+2+3), P73~P77 (rrnB from _C. fimi_ genomic DNA) (15.08.08: AM)
  • Gel 55:* M144-M147 (pSB1A2+cenA), M148-M151 (pSB1A2+cex) and M152-M155 (pSB1A2+dxs+crtE) digested with EcoRI/PstI. (18.08.08: AH)
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