Edinburgh/Week 15

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Week 15 (22~28 September 08)


  • Clones for ISA1/zm1 (M229) and ISA2/zm2 (M230, 231, 232) obtained.
    • These have the internal EcoRI as expected so they are probably the right genes but should be sequenced to confirm that the ends are intact.
    • M229 (ISA1) and M231 (ISA2) submitted for sequencing. (CF)
  • Clones for cex mutant (M233, 234, 235) obtained.
    • These clearly do not have the internal PstI site.
    • M233 submitted for sequencing. (CF)
  • Candidate clones for cenA (M237, 238) obtained.
    • These seem to possess the expected internal SalI sites.
    • One of these has been submitted for sequencing. (CF)
  • Candidate clones for bglX (M240, 247) obtained.
    • Submitted for sequencing but not so confident about these: the ratio of white to blue colonies was very low. There might be a problem with the restriction site in one of the primers.
  • rbs added to glgC and glgC16; cloned as M243 and M245. Both submitted for sequencing. (CF)
  • lac promoter (J33207) added to dxs+lims+appY Biobrick.
    • Does not smell of lemons but E. coli has a strong smell so this might not be significant.
    • Arranged for GC to be done to test for limonene production. (CF)
  • Ligation and transformation to add crtBI upstream of dxs+crtE. Minipreps to be done.
    • None of the patches are pink, but then, there is no promoter. (CF)
  • C. fimi genomic DNA submitted for sequencing. (CF)

Monday 22 September 08

  • Gel 201 (started new numbering as not sure what number we were up to): checking PCR products P91-P94 (ZmISA1 fusion PCR with and without rbs; ZmISA2 ditto) and P95, P96 (cex mutagenic PCR). P96 looks OK.
  • Purified P91 and self ligated = L68. Later P93 (L69) and P96 (L70).
  • Transformed L68, plates 205 and 206.
  • Digests of M168 and M212 (glgC and glgC16) to add rbs. Ligations L71, L72.
  • Minipreps M222-225 of Plac+dxs+LIMS+appY. Gel 202 shows digests, all look OK. Also on Gel 202 checked P65 (cenA) and P90 (bglX): both look OK.

Wednesday 24 September 08

  • Subbed possible Zm1 transformants from plates 205 and 206 to plate 207.
  • PCR with L71 and L72 as template to obtain rbs+glgC, rbs+glgC16 = P99, P100. Ran on gel 203, look OK.
  • Transformed JM109 with L69, L70 (ZmISA2 and cex mutagenic PCR self ligations). Plated to plates 208 to 211.
  • Digested P65 (cenA) and P90 (bglX) to insert into Edinbrick1. Ligations L73, L74.

Thursday 25 September 08

  • Digests to clone P99, P100 (rbs+glgC, rbs+glgC16) in Edinbrick1; ligations L75, L76.
  • Minipreps 226 to 229 of possible sob-ZmISA1 clones. Gel 204 of PstI digests shows that only 229 looks right; others hardly have any DNA at all.

Friday 26 September 08

  • Further digests of M229 (possible sob-ZmISA1) with EcoRI, EcoRI-PstI show that the internal EcoRI site is present, so probably insert is correct. (Gel 205)
  • Digests of X13 (crtBI) and X21 (dxscrtE) to insert crtBI upstream of dxscrtE. Size of crtBI insert band too close to vector to separate on gel (Gel 205). Set up ligation anyway (L77?).
  • Transformed L75, L76 (rbs+glgC, glgC16), plates 218-221.
  • Subbed possible cenA and bglX clones (v. few white colonies for bglX) to plate 217.
  • Minipreps M230-232 of possible sob-ZmISA2, and M233-235 of possible cex mutants. PstI digests of all 6 look good (gel 206), no internal PstI site in cex so mutagenesis successful (but need to sequence to confirm clean joining of ends, no frameshift mutation).

Saturday 27 September 08

  • Further digests of possible ZmISA2 and cex-mutant clones 230-235 (gel 207). All look good.
  • Transformed L77 (crtBI+dxscrtE), plates 222-223?
  • Minipreps M236-238 (cenA), 239-241 (bglX). EcoRI digests show M237 and M238 look OK for cenA, 240 looks possible for bglX, others definitely wrong (Gel 208).
  • Defrosted small freezer.

Sunday 28 September 08

  • Further digests of M237 and 238 (possible cenA) and 240 (possible bglX). EcoRI/PstI of M237, M238 look good, also internal SalI sites present. M240 is ambiguous at best (Gel 209).
  • Minipreps M242, 243 (rbs+glgC), 244, 245 (rbs+glgC16), 246, 247 (last 2 possible bglX white colonies). M243-245 look OK (M242 seems to lack SacI site so may not have rbs). No DNA in M246, but 247 is another possible bglX.