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Team:Edinburgh/Notebook
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Lab Notebook
Week 9
Monday 11 August 08
- PCR products of P55 to P58 (cenA and cex) were run on Gel 41: cex with KOD polymerase (P57) generated a single band of the desired size. However, cex with Velocity polymerase and cenA with both KOD or Velocity polymerase failed. (YAN)
- PCR products P61 to P63 (rrnB, cenA, cex) were run on Gel 42. Very clear band for rrnB, no bands for either cenA or cex. (CF)
- Made Plate 109/110 (Transformation of L33: pBS1A2+pCstA), 111/112 (transformation of L34: pBS1A2+dxs+crtE) and Plate 113/114 (Transformation of L35: pBS1A2+dxs+lims1). (Yan)
- Maxipreps X9 of pSB1A2+rbs+dxs (as for M72) and X10 of pSB1A2P+rbs+appY (as for M82). (AM)
- Redid PCR (KOD) of cex from C. fimi genomic DNA but with 10µl 50% glycerol, denaturing 1 min, annealing 60°C for 30s and extension 30s (P64). To be run on gel tomorrow. (AM)
- Digested P57 (cex from KOD PCR) with EcoRI/PstI to confirm identity of the gene. cex has a PstI restriction site near its midpoint, so this digestion should generate two segments of approximately 750bp. To be run on gel tomorrow. (AM)
- Digests performed using EcoRI/PstI to move crtE from BABEL2 (M79) to Edinbrick1. In each case, 3µl of vector was digested in 20µl total volume. The digests were run on a gel ready for excision, but gel showed no band corresponding to crtE (see gel 43 showing a single band at ~3kb for BABEL2+crtE and bands at ~2.5kb and 800bp for Edinbrick1). (AH)
- M109-M112 Minipreps of pSB1A2+crtB+crtI, from plates 102. M113-M116 Minipreps of pSB1A2+glgC-mut1,2 from plate 103. M117-M120 Minipreps of pSB1A2+glgC-mut1,2,3 from plate 94 (OG)
- Double digestion of M109 to M120 with EcoRI/PstI, run on gel 44. M110-102 (pSB1A2+crtB+crtI) have a band at 2.5kb as expected. M109 (pSB1A2+crtB+crtI) gave a band at 3kb instead, so should be ignored from now on. (Yan)
Tuesday 12 August 08
- Plate 115 made with subs from L33 (Pl. 109/110: pSB1A2+pCstA), L34 (Pl. 111/112: pSB1A2+dxs+crtE) and L35 (Pl. 113/114: pSB1A2+dxs+lims1) (CF)
- Over-night cultures of L33 (pSB1A2+pCstA, sub-cloned on Plate 115), L34 (pSB1A2+dxs+crtE, sub-cloned on Plate 115) and L35 (pSB1A2+dxs+lims1, subcloned on Plate 115) set up, ready for minipreps tomorrow.
- M121-M123: Minipreps of pSB1A2+crtY (1,2,3). M124-M126: Minipreps of pSB1A2+rbs+crtY (1,2,3). note: I messed up M121 by adding 200µl instead of 100µl of sol 1. They are in the green box, the orange one is full. (OG)
- Double digests of M121 to M126, run on Gel 46. It looks good! M121 is good! (Yan)
- Purified P61 (rrnB) for sequencing. (CF/AM)
- Re-purified already purified genomic DNA from C. fimi. This meaningless endeavour resulted from a misunderstanding. Hopefully, the doubly-purified DNA will still be useable. (AM)
- P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (su1) and P67 (iso2) run on Gel 45. P57 double digest yielded no clear bands; P64 yielded a clear band at 1.5 kb. (CF/AM)
- Purified P57 (cex from cell suspension) and P64 (cex from genomic DNA). Digested both with PstI. (AM)
- PCR for cenA from C. fimi genomic DNA (P65): denaturing 1 min, annealing 56°C for 20s, extension 30s, with glycerol. (AM)
- P57 and P64 (cex) PstI digests and P65 (cenA) run on Gel 47. Results indicate success!. (AM)
- Re-did the excise of crtE from BABEL2 (M79); cut out the band responsible for crtE and Edinbrick 1 on the gel, followed by putification of crtE and Edinbrick1 from the cut-out gel. Finally, ligation of crtE to Edinbrick1 (L38). (HX, AH)
- Analytical digests as follows (AH):
- M109 and M110 (pSB1A2+crtB+crtI): BamHI/EcoRV
- M115 and M116 (pSB1A2+glgC-mut1,2): HindIII
- M115 and M116 (pSB1A2+glgC-mut1,2): EcoRI/HindIII
- Digests of M109, M110, M115 and M116 were run on gel 48. M109 and M110 should give two bands if both genes are present. HindIII digest of M115/116 should give a single band indicating linearised plasmid with glgC. Double digest of M115/116 should give an insert band of ~1kb and a vector + ~500bp of insert band. Results do not look good. Bands seem to indicate much too large products, but will discuss with Chris tomorrow.(AH)
- M116 (pSB1A2+glgC-mut1,2) and M120 (BABEL2+glgC-mut1,2,3) submitted for sequencing. (CF)
Wednesday 13 August 08
- Submitted M109 (pSb1A2+crtB+crtI) for sequencing as AH_109F and AH_109R using primers pSB1A2insF2 and pSB1A2insR2 respectively. (AH)
- M127-M130: Minipreps of pSB1A2+pCstA (1,2,3,4), M131-M134: Minipreps of pSB1A2+dxs+crtE (1,2,3,4), M135-M138: Minipreps of pSB1A2+dxs+lims1 (1,2,3,4). All from plate 115. Note: M132 and M136 might be labelled wrong, the ethanol erased the writing on the tubes. Sorry! (OG)
- Double digests performed on M127-138 using EcoRI/PstI. (AH, OG)
- Transformation of L38 (pSB1A2-crtE) on Plate 116 (100ml) and plate 117 (900ml). (HX)
- Ligation of P54 (cex) and P65 (cenA) into Edinbrick1, to make L39 (pSB1A2+cex) and L40 (pSB1A2+cenA). (Yan)
- PCR for cenB (P68) and cenC (P69) from C. fimi genomic DNA: denaturing 1 min, annealing 56°C for 20s, extension 85s, with glycerol. Run on Gel 49. Results indicate failure. (AM)
h5. Thursday 14 August 08
- Subplated from plate 116 (pSB1A2+crtE 100 microlitres) onto *plate 118*. Should be ready to make culture for miniprep later this afternoon. (AH)
- Submitted M121 (pSB1A2+crtY), M124 (pSB1A2+rbs+crtY), M130 (pSB1A2+pCstA) and M137 (pSB1A2+dxs+lims) for sequencing using pSB1A2insF2 for sequencing of all 4 + pSB1A2insR2 for M137. Also resubmitted M109 (pSB1A2+crtBI) for sequencing using both forward and reverse primers, because I realised that yesterday I circled BigDye rather than "reaction required" on the sequencing form\! (AH)
- Made culture of pSB1A2+CrtE from plate 118, which was subcloned from plate 116 for minipreps (1-4) (YAN & HX).
- Transformation of L39 (Cex+Edinbrick1) into *plate 121/122;* and L40 (CenA+Edinbrick1) into *Plate 119/120.* (YAN).
- Digested and ligated M63 (crtE) and M72 (dxs). for M63 i used buffer H, XbaI, and PstI, For M72 i used buffer B, SpeI and PstI. M63 in inserted downstream of M72. the DNA samples of M63 and M72 were taken from the gel after using SYBR-Safe staining. (OG)
- Purified four preps of _C. fimi_ genomic DNA from bottled cultures made by Dr French (in LB, made from _C. fimi_ NA plates on lab bench). (AM)
- PCR of M43 (glgC mut 1+2 -->- *{-}P70{-}*\-) and M120 (glgC mut 1+2+3 \--> *P71*) with blunt-ended glgC primers. Run on *Gel 52* (?) . Results: P70 failed; P71 yielded a thick band around 1.5kb (proper size for glgC) and two unexpected bands around 6kb and 12kb. (AM)
h5. Friday 15 August 08
- Plates from yesterdays transformations of pSB1A2\+_cex_ and pSB1A2\+_cenA_ (119-122) failed to show any signs of growth. The transformation's being repeated. (AH)
- Glycogen assay performed: Flooding colonies with Gram's iodine solution yields results after \~5 minutes. Colonies on the (+)glucose plate stained darker than those on the (-)glucose plate (brown compared to no apparent change). The staining seems to grow more apparent up to \~30 minutes and then fades to nothing as the plate dries off. (AH)
- Transform with L39(cex+Edi), L40(cenA+Edi) & L41(dxs/crtE) on Plate123 to 128.(HX)
- Minipreps of pSB1A2+CrtE from its corresponding culture 1 to 4, made *M139 to M143*. (M142 and M143 made from the same culture (4). M143 has double quantity of solution 1.) Then, double digeats of M139 to M143 with EcoRI and PstI and the products were run on *Gel 53*. (YAN/OG)
- PCR of M43 (repeat: glgC mut 1+2 -->- *{-}P71A{-}*\-), M120 (repeat: glgC mut 1+2+3 \--> *P72*), and rrnB from the four _C. fimi_ genomic DNA preps made yesterday plus the original prep as a positive control (*P73~77*). PCR products run on *Gel 54*. Results: P71A and P72 succeeded, producing large quantities of DNA around the length of glgC; P74 yielded a single band around 1.5~2 kbp whilst P73 and P75 yielded gel-length smears and nothing came of P76 and P77 (the control). (AM)
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
