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Team:Edinburgh/Notebook
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Lab Notebook
Week 5
Monday 14 July 08
- Digests and ligation L12 to add rbs to appY clone M15 (sequence back today, seems OK).
- Minipreps M31 to M42 of crtB and crtI clones. Digests with EcoRI/PstI. (AM)
Tuesday 15 July 08
- PCR P15: fusion PCR of rbs+appY using L15 as template. Extension 20s, annealing 58°C, same primer set as before. Gel 13 shows that it worked OK.
- Gel 13 also shows digests of M31 to M42 from yesterday.
- Transformation of JM109 with ligations L13 and L15 (glgC mutation and rbs+dxs cloning). Plated to plates 39 to 41.
Wednesday 16 July 08
Meeting with other UK iGEM teams.
Thursday 17 July 08
- PCR for cenA, cenB, cenC and cex (P16, P17, P18, P19). Annealing 58°C, extension 115s (cenB and cenC are both over 3kb) and included 5% v/v glycerol in the reactions since in the past that has been helpful with high GC templates. PCR products purified to be run on gel tomorrow (against wisdom, since they should have been run on gel both before and after purification).
- Results of transformations: plenty of colonies for glgC mutation (mutating site 1 in the mutant which already has site 2 mutated). Subbed some of these to plates 42, 44 and 45 (poor communications). Only a few colonies for rbs+dxs, and only one of these was white. Subbed this to plate 43. Apparently there may have been some problem with the purification of this digest - may need to repeat.
- Re-digested M32, M36 (pSB1A2-crtB), M39 and M40 (pSB1A2-crtI) and ran on Gel 15. Gel unsuccessful - stain smeared over 0.5-2kb region, exactly where crtB and crtI bands are expected to appear. Perhaps too much loading buffer or problem with the tank (tank with 'loose wire' was used). (AM)
- pSB1A2+appY cultures set up in beaker for maxiprep tomorrow (X3). (AM)
Friday 18 July 08
- Made pSB1A2+appY Maxiprep *X3*. (AM)
- Purified PCR products for cenA, cenB, cenC and cex (P16, P17, P18, P19) run on Gel 16. No visible bands. (AM)
- Second attempt at PCR for cenA, cenB, cenC and cex (P20, P21, P22, P23). Annealing 55°C, extension 85s; did not include glycerol. Ran unpurified PCR products on Gel 17 - no bands apart from thick bands in the <0.5kb region = primers. Hence, PCR was unsuccessful. (AM)
Saturday 19 July 08
- Minipreps M43 to M47 of possible glgC double mutants (both EcoRI sites gone), and M48 of the only white colony from the pSB1A2+rbs+dxs transformation. Gel 18 shows EcoRI/PstI digests of 3μl. M43 to M47 all look about right: send one for sequencing. M48 is ambiguous, could be incomplete deigestion, need to repeat. (CF)
- PCR P24 and P25, repeat PCR for cenA and cex (the two shorter genes) using Pfu polymerase instead of KOD, adding 5% glycerol - this worked well for high-GC genes from Saccharothrix espanaensis last year. (CF)
Sunday 20 July 08
- Ran P24 and P25 on Gel 19. No amplification products. Time to do some trouble shooting. The fault is in either the primers, the template, or the reactions. All previous reactions have been fine, so most likely the primers or template. To check this, we need to do a positive control using universal primers such as fd1 and rd1 (amplify 16S rRNA gene rrnB) which should definitely give a product. If these fail, then it is most likely that the template is at fault - we should prepare genomic DNA and try using this as template rather than cells; also do a Gram stain to confirm that the cells look OK. If the control reaction works, try remaking the primer solutions, though it seems unlikely that all four sets could be wrong, unless I was having a really bad day. (CF)
- Redid digest of M48 with SacI/Spe1. Does not look good. Also repeated digests of crtB and crtI clones M36 and M42; alternating single digests (EcoRI) and double digests (EcoRI/PstI) to check for inserts. Look OK. Submit for sequencing. (CF)
- Also: I have a plan for in vitro assembly to cut down on transformations and minipreps. Could have a try of this next week if there are some students with some free time. (CF)
Week 6
Monday 21 July 08
- M36 (crtB), M42 (crtI) and M43 (glgC-mut1,2) submitted for sequencing. (AH)
- Plates 46 and 46 have no colonies (white or blue) - Something went wrong with the transformation. (AH)
Tuesday 22 July 08
- Positive control PCR with universal primers fd1 and rd1 to amplify 16S rRNA gene rrnB from C. fimi (P26); PCR products run on Gel 21 - failed. (AM)
- Made overnight culture of C. fimi from plate 37 for chromosomal DNA purification tomorrow. (AM)
- M36 (crtB) and M42 (crtI) double digested, ligated with rbs to make L17 (M36) and L18 (M42). (YAN & OG)
- L15 (rbs+dxs ligation to Edinbrick1) and L16 (rbs+appY ligation to Edinbrick1) run on gel, the result showed the ligations into edinbrick1 failed. (YAN & OG)
Wednesday 23 July 08
- M43 (glgC-mut1,2) sequence results received. First and second mutation sites successfully removed. (AH)
- Overnight culture of C. fimi did not yield sufficient cells. Made two more cultures in nutrient broth; all cultures incubated at 30°C with shaking. (AM)
- PCR of crtY from P. ananatis cells (P27) and crtB and crtI from ligations L17 and L18 respectively (P28, P29). PCR products were run on Gel 22 ~ P28 and P29 successful, P27 unsuccessful. (AM, YZJ, OG)
- Re-did positive control PCR for rrnB from C. fimi (P30) ~ denaturing at 95°C for 20s, annealing at 55°C for 1 minute, extension at 70°C for 30s. The first two settings were in error; denaturing should have been set to 1 minute and annealing to 10 sec as usual. HOWEVER, annealing for 1 minute appears to have worked, so it might be worth trying this for PCRing the cellulases. (AM)
- P30 (the above) and pZntA were run on Gel 23. Both successful. (AM)
Thursday 24 July 08
- Purified PCR products P28 and P29 (OG)
- Double digestions using XbaI/PstI (buffer H) to add P12 (rbs+dxs), P15 (rbs+appY), P28 (rbs+crtB) and P29 (rbs+crtI) to Edinbrick1. (Yan, OG)
- Double digestion using SacI/SpeI (BufferE) to add P31 (pZntA) to Edinbrick1. (Yan, OG)
- Purified genomic DNA from C. fimi culture (made from plate 37, incubated in 25ml bottle at 30C for 48 hours with shaking). Used Promega Wizard Genomic DNA Purification Kit, lysozyme and RNAse+EDTA (as replacement for proprietary RNAse Solution, of which none was left). Final solution left overnight in refrigerator at 4°C. (AM)
- Plated C. fimi culture from plate 37...
- Incubated in 25ml bottle for 48 hours at 30°C with shaking ... to Plate 48.
- Incubated in 50ml bottle for 24 hours at 30°C with shaking ... to Plate 49.
- Incubated in 250ml flask for 24 hours at 30°C with shaking ... to Plate 50. (AM)
- PCR of cenA (P32), cenB (P33), cenC (P34), cex (P35), annealing 1 min 55°C, extension 90 sec 70°C. Run on Gel 24 ~ unsuccessful. (AM)
Friday 25 July 08
- Made maxiprep X4 (pSB1A2-crtB as M36; BBa_K118002) and X5 (pSB1A2-crtI as M42; BBa_K118003) (AH, XH)
- Ligation of P29 into Edinbrick1 to make L22 (pSB1A2+rbs+crtI). (Yan, OG)
- Ligation of P28 into Edinbrick1 to make L21 (pSB1A2+rbs+crtB). (Yan, OG)
- Ligation of P12 into Edinbrick1 to make L19 (pSB1A2+rbs+dxs). (Yan, OG)
- Ligation of P15 into Edinbrick1 to make L20 (pSB1A2+rbs+appY). (Yan, OG)
- Ligation of P31 into Edinbrick1 to make L23 (pSB1A2+pZntA). (Yan, OG)
- C. fimi Plate 48 appears to be contaminated (mix of opaque and clear colonies). The same culture was used for genomic DNA purification yesterday ~ this preparation is therefore inadequate for sequencing purpose but might be worth using for PCR. (AM)
- No growth of C. fimi on Plates 49 and 50, but C. fimi takes a relatively long time to form colonies so we should check again on Monday. (AM)
- PCR P36 of M43 (glgC-mut1,2) to excise the gene from BABEL and rectify its orientation and P37 of crtY from P. ananatis (Plate 17) cell suspension - repeat of P27.
- P36 and P37 run on Gel 25. P36 tentatively successful (larger than desirable smear in the region of interest), P37 failed.
- Also did preparations to transfer crtE from BABEL2 vector to pSB1A2. This is necessary, because we are supposed to use Registry standard vectors for all BioBricks we make (in fact, we should even stop using pSB1A2and move to pSB1A3). So, digested the BABEL2+crtE clone that Douglas Leslie made with EcoRI and PstI, and also Edinbrick1 - in both cases, 3μl in 20μl total volume. Ran both on a gel and excised the appropriate bands, eluted each to 10μl EB and set up a ligation using 4μl of each in 10μl total volume. Ligated at 16°C. (CF)
Saturday 26 July 08
- Transformed JM109 with 5μl of ligations labelled as L12, L15, L28, L29, L31 (note: these ligations seem to have been numbered based on the PCR product they were derived from rather than sequentially; they have been renumbered as L19 to L23), plus my own ligation (L24) which was to transfer crtE from the BABEL vector (which is not an 'official' BioBrick vector, though it is fully compliant) to pSB1A2, so that we can submit it to the Registry. These were plated on plates 51 to 62. (CF)
Sunday 27 July 08
- Result of transformations: no growth for 'L12' (L19), 'L15' (L20) or 'L28' (L21). For 'L29' (L22) there are some blue and white colonies. For 'L31' (L23) there are many blue colonies but only a few white ones, suggesting that the SacI/SpeI double digest is less efficient than the XbaI/PstI which was (I hope) used for the others. For the crtE ligation, there are many white colonies and very few blue - this is as expected, since in this case the vector and insert bands were purified from a gel. (CF)
Week 7
Monday 28 July 08
- Repeated transformation of L20 (pSB1A2+rbs+appY) and L21 (pSB1A2+rbs+crtB), and plated on plates 63 to 66. (YAN and Omar).
- PCR of cenA, cenB, cenC and cex from cell suspension (P38-41; cell solution incubated at 73-80°C for 8 minutes to kill cells/inactivate DNAse) and DNA solution 'purified' from contaminated cell culture (P42-45). Denaturing 95°C for 1 min, annealing 55°C for 10s, extension 70°C for 90s. PCR products run on Gel 26. (AM)
- Prepared L25 of P36 (glgC-mut1,2 PCRed with primers f2 and r2) and Edinbrick 1. (AM)
- Minipreps M49 to M54 of pSB1A2+rbs+crtI transformants. (CF)
Tuesday 29 July 08
- Transformation of L20 and L21 failed.
- Transformation of glgC-mut1,2 ligation product, made plate 67 & 68 (Yan)
- Cut out putative cenA and cex (both ~1.5kb) fragments from gel 27 (repeat of Gel 26 but stained with SYBRsafe). Purified DNA from gel fragments: F1 (putative cenA) and F2 (putative cex). (AM)
- Digests of M49 to M54 (pSB1A2+rbs+crtI) with EcoRI alone. This should generate a single 4 kb band (doing an EcoRI/PstI double digest would give a vector and insert band both around 2 kb, which would probably run at the same place, making it hard to tell whether there was an insert). These were run on Gel 28. All showed a single band at the expected place. (CF)
- Minipreps of pSB1A2+pZntA (M55 to M60) from L23 (Yan, Nimisha)
- Minipreps of pSB1A2+rbs+crtE (M61 to M66) from L22 (Yan, Nimisha)
- Double digests of M55 to M60 and M61 to M66 with EcoRI/PstI. This should generate the following bands: ~2.2 kb for pZntA (M55 to M60), ~1.1 kb for rbs+crtE (M61 to M66), and ~2 kb for the pSB1A2 vector. These were run on Gel 29. M55 to M60 unsuccessful; M61 to M66 showed a band at the expected location for rbs+crtE but seemed to be indicate that L22 was still ligated into BABEL2 instead of pSB1A2. (Yan & AM)
Wednesday 30 July 08
- P48 (crtY) and P49 (Recreation of P12, rbs+dxs) created (30.07.08: CF)
- Ligated F1 (putative cenA) and F2 (putative cex) to Edinbrick 1 (L28 and L29 respectively). All were digested with EcoRI/SpeI, purified and stored overnight in the 16°C water bath. (AM)
- Repeated PCR for cenA and cex (P46, P47) from heat killed cell solution. Denaturing 95°C for 1 min, annealing 65°C for 10s, extension 70°C for 40s. Run on Gel 30. Unsuccessful, perhaps because annealing temperature was too high (temperature was decided based on stock solution label, which took the prefix/suffix into consideration). (AM)
- Gel 31: M49 and M50 (pSB1A2-rbs+crtI digested with a) XbaI, b) SpeI/XbaI, c) sac/speI), M63 (BABEL2+rbs+crtE digested with a) EcoRI, b) EcoRI/PstI), M67 (pSB1A2+rbs+crtB digested with a) EcoRI/PstI, b) sacI/speI) and P48 (crtY). (OG)
- Re-digested M55 to M60 (pSB1A2+pZntA) with a) EcoRI and b) EcoRI/PstI. (This time, the amount of DNA for digestion is increased). (Yan)
- Gel 32 results look promising for M57, but not for the others. (Yan)
- Purified P15 (rbs+appY) and P28 (crtB), digested each with EdinbrickI using XbaI/PstI and set-up for ligations (L26 and L27). Stored overnight in the 16°C water bath.(HX)
Thursday 31 July 08
- M50 (pSB1A2+rbs+crtI), M63 (BABEL2+rbs+crtE) and M67 (rbs+crtB) submitted for sequencing. (CF)
- Transformation of L26 (pSB1A2+rbs+appY) to Plates 75 and 76, L27 (pSB1A2+crtB) to Plates 77 and 78, L28 (pSB1A2+cenA) to plates 79 and 80 and L29 (pSB1A2+cex) to plates 81 and 82. (HX)
- Minipreps of M68-M71 (pSB1A2+glgC-mut1,2), M72-M75 (pSB1A2+rbs+dxs) and *M76-M79* (BABEL2+crtE). (YAN, AM)
Friday 1 August 08
- Primers for pCstA ordered. (CF)
- White colonies from Thursday's transformations were subplated as follows...
- Plate 76 (L26: pSB1A2+rbs+appY) to Plate 83
- Plate 77 (L27: pSB1A2+rbs+crtB) to Plate 84
- Plate 80 (L28: pSB1A2+cenA) to Plate 85
- Plates 81/82 (L29: pSB1A2+cex) to Plate 86. (AM/HX/OG)
- First digestion: M68 to M71 (pSB1A2+glgC-mut1,2) with EcoRI/PstI, M72 to M75 (pSB1A2+rbs+dxs) with EcoRI, M76 to M79 (BABEL2+crtE) with EcoRI/PstI. Run on Gel 33. Results: M68, 69, 71: vector band around 2 kb, no glgC band; M72, 73, 74: single band around 4 kb, expected length for vector+gene; M76-79: crtE band <1 kb, vector band ~2 kb. It is not clear into which vector crtE (M76~79) is inserted, so sequencing will be necessary.
- Second digestion: M68 and M69 with a) EcoRI, b) EcoRI/PstI; M72 with a) EcoRI b) SacI/SpeI. Run on Gel 34. Results: Single and double digests of M68 and M69 yielded similar results, hence pSB1A2+glgC-mut1,2 ligation or transformation failed; digests of M72 yielded expected bands, hence pSB1A2+rbs+dxs successful. (AM)
- P36 (glgC-mut1,2 excision from BABEL2) and L25 (P36 + Edinbrick1) were run again on Gel 35. Results: P36 viewed at lower illumination yields one band of the desired length (1.5kb); L25 yields two bands around 1.5kb, a thick band at 3kb and several fainter bands including one at 2kb (length of pSB1A2). (AM)
Week 8
Monday 4 August 08
- Made 17 minipreps from transformations of L26, L27, L28, L29: M80-83 (L26: rbs+appY in Edinbrick1), M84-85 (L27: rbs+crtB in Edinbrick1), M86-90 (L28: cenA in Edinbrick1) and M91-96 (L29: cex in Edinbrick1). This was followed by double digestion of all the miniprep products (M80-96) with EcoRI/PstI. (Yan, OG)
- Third glgC mutagenesis made by MABEL on BABEL2+glgC-mut1,2 (since pSB1A2+glgC-mut1,2 appears to have failed) performed: P50 (Yan)
- Double digestions of M80 to M96 and P50 were run on gel 36. Results were good for M80-82 (pSB1A2+rbs+appY), M84-85 (pSB1A2+rbs+crtB), M92 (pSB1A2+cex) and P50 (glgC-mut1,2,3). Other results were poor, with bands in the wrong position for M86-M90 (pSB1A2+cenA). (Yan, OG)
- Sequencing results back today indicate that M50 = pSB1A2+rbs+crtI, M63 = pSB1A2+rbs+crtE and M67 = pSB1A2+rbs+crtB. (AH)
Tuesday 5 August 08
- M72 (pSB1A2+rbs+dxs), M79 (BABEL2+crtE) and M82 (pSB1A2+rbs+appY) submitted for seqencing. (CF)
- Minipreps M97-104 of pSB1A2 cenA (2, 7-13) and M105-108 Minipreps M105-108 of pSB1A2+cex. (Yan)
- M97-108 were double-digested with EcoRI/PstI and run on gel 37. (Yan)
- Purification and self-ligation of P50 (3rd mutagenesis of glgc-mut1,2) (L30). (HX)
- M68-69 (glgc-mut1,2 in pSB1A2) and M92 (pSB1A2+cex) were digested with a) PstI and b) EcoRI/PstI, but gel leaked. Entire process needs repeating tomorrow! (CF, OG)
- Digested P36 (glgC-mut1,2) with Edinbrick1 using Spe1/EcoR1 - to be continued tomorrow. (OG)
- Plate 93 created from subculture of plate 56 (M67, pSB1A2+rbs+crtB)
Wednesday 6 August 08
- L31 ligation of P36 (glgC-mut1,2, hopefully, could be P39) to Edinbrick1 completed. (OG)
- Primers have arrived for pCstA. Chris carried out the PCR (that would be P51). Unsuccessful. (CF)
- Digestion of M67 (pSB1A2+rbs+crtB) with EcoRI/SpeI and M50 (pSB1A2+rbs+crtI) with EcoRI/XbaI, in preparation for combining the two with the former as insert and the latter as vector. (CF)
- Not enough time to purify/ligate due to high demand for agarose gel following lunchtime. Shall do tomorrow morning at 9. (AM)
- Re-digestion of M97, M98, M99 and M101 (all pSB1A2+cenA) and M105 and M108 (pSB1A2+cex) with a) EcoRI, b) EcoRI/PstI, and M92 (pSB1A2+cex) with a) PstI, b) EcoRI/PstI. All run on gel 38 (because gel 37 did not look good). (Yan)
- Transformation of L30 (Self-ligation of BABEL2+glgC-mut1,2,3) (plates 89 and 90) and L31 (glgC-mut1,2 to Edinbrick1) (plates 91 and 92) onto B/W plates. (HX)
- Maxipreps X6 (as M50, pSB1A2+rbs+crtI) and X7 (as M63: pSB1A2-+rbs+crtE) made. (AM)
- Overnight culture made from plate 93 (pSB1A2+rbs+crtB) ready for maxiprep tomorrow. (AM, AH)
- Analytical digests using a) EcoRI, b) EcoRI/PstI of M70, M71 (both pSB1A2+glgC-mut1,2) and M92 (pSB1A2
+cex). Run on gel 39. Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (CF)
Thursday 7 August 08
- Subcultured from Plates 89 and 90 (BABEL2+glgC-mut1,2,3) to Plate 94. Plate 91 (pSB1A2+glgC-mut1,2) showed no growth, so was discarded. Plate 92 (pSB1A2+glgC-mut1,2) had a blue smear and two white colonies. These colonies were subbed to Plate 95. (HX)
- Maxiprep X8 (as M67: pSB1A2+rbs+crtB) made. Slight hiccup after step 6. (Cold solution 3 was added and incubation on ice was skipped. Centrifugation continued for 2 minutes before mistake was realised, after which the solution was mixed and incubated on ice for ten minutes (continuing with step 7). (AM, AH)
- Combination (Ligation) of rbs+crtB (from M67) and rbs+crtI (from M50): rbs+crtB as an insert cut with EcoRI/SpeI; rbs+crtI as a vector cut with EcoRI/XbaI (L32). (Yan, AM)
- Double digestion of three biobricks: 2 of rbs+dxs (from M72), rbs+crtE (from M63) and rbs+lims1 (from 0713536 in iGEM 07 box): rbs+dxs as a vector cut with SpeI/PstI; rbs+crtE as an insert cut with XbaI/PstI; rbs+lims1 as an insert cut with XbaI/PstI.
- Made PCR of cenA and cex again, stored in P52 (cenA) and P53 (cex). The PCR products were checked on gel, but failed again! (Yan, AM)
- PCR P54 of pCstA. Run on gel 40. Results look promising (fairly prominent band <500bp). Purified. (CF)
- Plates made for testing glycogen assay. 2x LB agar, ampicillin; 2x LB agar, ampicillin, 2% glucose ready for spreading with E. coli. (SK)
- Plates 96 and 97 (glycogen assay) were spread with subcultures from Plate 43. Plate 96 = (-)glucose, plate 97 = (+)glucose. (HX)
Friday 8 August 08
- Plate 94 (BABEL2+glgC-mut1,2,3) has white cultures, so can be prepped for minipreps. Plate 95 (pSB1A2+glgC-mut1,2) has blue cultures. Therefore transformation of L31 (glgC-mut1,2 to Edinbrick1) has been repeated on plates 100 and 101. (HX)
- Transformation of L32 (pSB1A2+crtB+crtI) on plates 98 and 99. (HX)
- Digested pCstA (P54) and Edinbrick1 using Spe1/EcoR1. Ligated as L33 to be transformed tomorrow (OG)
- Arabidopsis isa1 and isa2 cDNAs have arrived as well as Zea mays su1, iso2 and iso3. The Arabidopsis genes are full of forbidden restriction sites, so it would be best to use the maize genes. Primers will need to be designed. (AH)
- PCRed cenA and cex with a) KOD polymerase, b) Velocity polymerase separately to make P55 (cenA by KOD), P56 (cenA by Velocity), P57 (cex by KOD) and P58 (cex by Velocity). (Yan)
- Combined rbs+dxs with rbs+crtE as L34, and rbs+dxs with rbs+lims1 as L35. L34 and L35 ligation is being allowed to occur at 16°C overnight. (Yan)
- M79 sequencing results indicate that M79 is BABEL2+crtE. (AH)
Sunday 10 August 08
- Extracted C. fimi genomic DNA, followed by PCR of rrnB (P61), cenA (P62) and cex (P63) from genomic DNA. (CF)
- Made subcultures...
- Patches from Plate 99 (L32, crtB+crtI) to Plate 102.
- Patches from Plate 101 (L31, glgC double mutant+pSB1A2) to Plate 103.
- Patches from Plates 105 (pSB1A2+crtY) and 106 (pSB1A2+rbs+crtY) to Plate 108. (CF)
- Transformed competent cells with L36 pSB1A2+crtY (Plates 104/105) and L37 pSB1A2+rbs+crtY (Plates 106/107). (CF)
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
