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=== The Experiments === | === The Experiments === | ||
Revision as of 14:37, 1 July 2008
Contents |
University of Edinburgh 2008 iGEM Team
Team Members:
Students:
Andrew Hall, Adler Ma, Antonia Mayer, Omar Gammoh, Wenhong (Tina) Li, Xing (Ariel) He, Zejun Yan.
Main instructors:
Chris French, Alistair Elfick, Hongwu Ma
Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs)
| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook |
|---|
(Or you can choose different headings. But you must have a team page, a project page, and a notebook page.)
Saving the World
"Cellulose is the most abundant form of fixed carbon, with 100,000,000,000 tons produced in cell walls by plants each year." [http://www.blackwell-synergy.com/doi/full/10.1196/annals.1419.026 (Wilson, 2008)]
Nowadays the world-wide food shortage is becoming more and more important. As we can image, converting cellulose to starch will be one of the most sufficient ways to solve this problem .
Project summary
Recent Wiki updates
01 July 2008
Lysis: An alternative to cellulase secretion page updated. (AH)
The Experiments
Labwork Summary
- Entries up to 01.07.2008 copied from Dr French's wiki
iGEM 2008 Labwork Summary
Tuesday 17 June 08
Prepared competent JM109 cells using TSS method. 25 tubes of 0.2 ml were prepared, labelled 'iGEM 17-6-8 Jnn' where nn is a number from 01 to 25. These are in the pink box in Garry's -80 C freezer. Thursday 19 June 08 Eluted DNA for BioBrick E0240 (GFP) from square 1001, well 4B and used this to transform half of tube J25; other half was transformed with 1.5 microlitres of J33207 (Edinbrick1) as a control to test the competence of the cells. Plated 100 microlitres of each to LA+amb+BW. Incubate at 37 C overnight. Plate 1: JM109 transformation with E0240 eluted DNA. Plate 2: JM109 transformation with J33207 plasmid DNA (control).
Friday 20 June 08 Result of transformation: the positive control was highly successful, with hundreds of colonies, but no colonies were present on the plate transformed with the BioBrick DNA indicating some problem with the DNA elution. Need to check that we got the method right. Ordered primers for dxs, appY and glgC. There are no forbidden restriction sites in the first two, so primers with full prefix and partial suffix were ordered to clone as EcoRI-SpeI fragments, but glgC has two EcoRI sites which will have to be mutated out. We therefore had a choice of cloning it initially as a XbaI-PstI fragment, or attempting the BABEL system, and decided to try BABEL first. If it proves too unreliable, we will need to order new primers to clone it the old-fashioned way. Primer dxsf1: gat gaattc gcggccgc t *tctaga+ tg agt ttt gat att gcc Primer dxsr1: gc t actagt a tt a tta tgc cag cca ggc ctt g Primer appyf1: gat gaattc gcggccgc t tctaga tg gat tat gtt tgc tcc Primer appyr1: gct actagt a tta tt a gtc aat tgt ttt gtt tat tcc Primer glgcf1: atg gtt agt tta gag aag aac gat c Primer glgcr1: tta tta tcg ctc ctg ttt atg ccc taa c
Tuesday 24 June 08 Sarah S wants to revive one of the BioBricks from the Registry (pBad-GFP), so we'll see whether she can get it to work. Wednesday 25 June 08 Primers arrived. Made up 500 uM stock solutions in EB and 10 uM working solutions (f and r together) in water. Performed PCR with Kod, annealing at 55 C and extending for 38 seconds (expected sizes are: dxs 1862+prefix and suffix; appY 749+prefix and suffix; glgC 1295 with no prefix or suffix (except the extra TAA). Result: all looked good, nice pure bands. Gel 1: markers, empty lane, dsx, appY, glgC, appY (repeat), glgC (repeat), markers.
Thursday 26 June 08 Purified DNA from PCR reactions. Used 20 microlitres of glass beads and eluted to 40 microlitres of TE. Sample P1: dsx PCR product Sample P2: appY PCR product Sample P3: glgC PCR product
Set up digests to clone appY and dxs in Edinbrick1. Digests with 32 microlitres water, 5 microlitres buffer E, 4 microlitres Edinbrick1 DNA, 4 microlitres purified PCR product, 2.5 microlitres SpeI, 2.5 microlitres EcoRI. Incubated at 37 C. Purified. Set up ligations: Ligation L1: dsx + Edinbrick1, E/S Ligation L2: appY + Edinbrick1, E/S Ligation L3: glgC (5 ul) +linear Babel1 (16-2-8, 2 ul) with PNK Ligation L4: glgC (5 ul) +linear Babel2 (18-2-8, 2 ul) with PNK
Incubate ligations at 16 C overnight.
Friday 27 June 08 Ordered Cellulomonas fimi ATCC 484 (NCIB 8980, DSM 20113) from DSMZ. Transformations of the iGEM competent JM109 cells with ligations L1 to L4. In each case, 100 microlitres of cells were transformed with 5 microlitres of ligation, and the remaining 5 microlitres of each ligation was frozen so that it could be analysed if the transformations fail. Fresh Blue-White Ampicillin plates were prepared. Plate 3: Ligation L1, 100 microlitres Plate 4: Ligation L2, 100 microlitres Plate 5: Ligation L3, 100 microlitres Plate 6: Ligation L4, 100 microlitres Plate 7: Ligation L1, 900 microlitres Plate 8: Ligation L2, 900 microlitres Plate 9: Ligation L3, 900 microlitres Plate 10: Ligation L4, 900 microlitres
Saturday 28 June 08 Result of transformations: Plate 3: 1 white; Plate 7: 5 white, 13 blue. Plate 4: no growth; Plate 8: no growth. Plate 5: 1 white; Plate 9: 3 white, 20 blue, possible signs of phage. Plate 6: maybe one tiny white; Plate 10: 7 white, 9 blue.
The white colonies were transferred to fresh plates of the same medium:
Plate 11: possible Edinbrick-dxs transformants. Plate 12: possible Babel1-glgC transformants. Plate 13: possible Babel2-glgC transformants.
The new plates were incubated at 37 C. The old plates were left at room temperature to see if any further colonies would appear.
Sunday 29 June 08 Streaks on plate 12 show strong signs of phage infection and are unusable apart from the single white one from plate 5. At least one of the streaks on plate 13 also has a couple of plaques, but plate 11 looks fine. This suggests that phage may have come from the Babel DNA stock (or the PNK, which seems unlikely). Set up overnight cultures (2.5 ml LB in 20 ml bijoux) for minipreps. Numbers 1 to 6 are Edinbrick-dxs from plate 11, all white, number 7 is from plate 12 (the white clone from plate 5), and 8 to 12 are from plate 13, white or pale blue. Incubated at 37 C with shaking. Also note: a couple more colonies turned up on plates 5 and 6. The one on 6 looks like an escape, but the one on 5 looks OK. Subbed them both to a fresh plate (Plate 14). Previous plates, apart from plates 4 and 8 which had no growth, were transferred to the cold storage room. Monday 30 June 08 Plasmid DNA minipreps: M1 to M6: Edinbrick-dxs white colonies M7: Babel1-glgC white colony M8 to M12: Babel2-glgC white and pale blue colonies
Minipreps M1 to M6 were digested with EcoRI and PstI (Gel 2). The expected pattern is pSB1A2 vector band at 2.04 kb (2079 bp less prefix and suffix) and dxs insert band around 1.9 kb. M1 showed bands around 1.1 and 2.1 kb. M5 showed no DNA. The other four showed a vector-like band around 2 kb and a fainter, fuzzier band around 3 kb, possibly a 'ghost' band. Thus none of the clones show the expected pattern of bands (although it is conceivable, since the vector and insert bands are so close in size, that they may be lying on top of each other; dxs has an internal EcoRV site at +504 and two HindIII sites at +606 and +1230, whereas pSB1A2 lacks such sites so this could be used to check). In conjunction with the total lack of growth on the appY plates, this suggests that something went wrong in the cloning procedure. The next step is to run the remaining ligation material on a gel and see what it looks like.
Tuesday 1 July 2008 Gel 3: minipreps M2, M3, M4 and M6 (pSB1A2-dxs clones) digested with EcoRI alone; lanes 5 and 6, 2.5 microlitres of ligations L1 and L2. Result: all 4 minipreps now give a 4.2 kb band (plus the same 3.2 kb 'ghost' band as before) consistent with pSB1A2 carrying a 2 kb insert. Would be nice to confirm identity using HindIII (2 internal sites). Both ligations show clear signs of insert and vector bands (oddly, the dsx insert band overlaps the pSB1A2 vector band whereas the appY insert band overlaps the lacZ' insert excised from Edinbrick1). However, no ligated bands are visible. Thus the DNA purification is fine; if there was a a problem, it is with the ligase or ligase buffer.
Gel 4: minipreps M7 to M12 (supposed to be glgC in Babel vectors) digested with EcoRI and PstI to excise the inserts. M10 and M11 have a single 3 kb band consistent with vector, but no insert band at 1.2 kb. M7, M8, M9 and M12 all show a single band at about 2.4 kb which is not consistent with Babel vectors if properly digested. Unless the digests totally failed, none of these plasmids would seem to contain glgC.
Primer Design
Use Artemis to find positions of start/stop codons and forbidden restriction sites (Start: All programs: School applications: Science and Engineering: Biological Sciences: Sanger: Artemis_v8) The primers should have a melting point of ~55°C (usually 17-18 bp), based on:
G/C = 4°C each A/T = 2°C each G/T = 0°C Internal mismatches complicate and can be -ve temps. Remember, dPol adds to the 3' end - don't get confused when only one strand is presented!
3' end of the primer should be G/C - This is a stronger bond than A/T, so it binds more strongly to the target DNA sequence and the Taq will extend the primer more reliably. General biobrick primers 5' part of the primer should be non-complementary (<~20bp) to the DNA being amplified, and this should contain the prefix and suffix. The sequence must start ATG and stop TAATAA - if this is not naturally the case, then replace the natural start/stop codons using non-complementary primer. It may be necessary to leave ~3 extra bases additionally at the 5' end of each primer as EcoRI (and other restriction enzymes?) won't cut at the end of sequences. Adding the extra bases basically fools EcoRI into thinking that its cutting in the middle of a DNA fragment. We should leave out one of the two 5' restriction sites to prevent annealing of the two primers to each other. (The restriction site will be added back in as part of the vector when the vector and insert are annealed together.) Therefore, prefix and suffix to use are: PREFIX Primer: gaattcgcggccgcttctag (if the following sequence is coding) SUFFIX Primer: tactagtagcggccgctgcag
Note, however, that the suffix is read for the 5'->3' strand (the same as the prefix), and therefore when designing the primer the complementary sequence must be used:
Sequence to use as suffix when designing the reverse primer: ctgcag cggccgc t actagt a
Mutagenic primers (for MABEL) (Designing mutant primers for Mutagenesis with blunt-end ligation (MABEL))
Forbidden restriction sites EcoRI - GAATTC NotI - GCGGCCGC XbaI - TCTAGA SpeI - ACTAGT PstI - CTGCAG
"Design two divergent non-overlapping primers, one forward and one reverse, centred on the offending restriction site. One of them obviously must include the base which is being mutated, preferably at or near the 5' end so that the mismatch does not affect annealing too much. The primers can be quite short (17 bases or so) since they don't overlap or include any non-complementary tails. The 5' ends of the primers must be adjacent to each other (on opposite strands, of course) so that the PCR product will include every base of the vector plus insert."
Our Primers
[http://www.openwetware.org/wiki/French_Lab Dr Chris French's OpenWetWare Site]
- Our one-stop destination for Biobrick protocols
External Links
[http://andhi.tiddlyspot.com/ Andy's lab book]
