Edinburgh/Week 16

Week 16 (29 September ~ 5 October 08)

lac promoter added to glgC and glgC16 biobricks, which were then grown overnight in LB and LB+40mM glucose.
- Cells were subsequently resuspended in iodine reagent to test glycogen levels - results are quite promising. See Results section.

xylE and lacZ reporter genes added to cstA promoter, then grown overnight in LB and LB+40mM glucose.
- xylE assays performed - results are very promising. See Results section.

cenA (M237~238) and cex (M233~235) biobricks confirmed. Ribosome binding sites are now being added.
bglX clones (M240, 247) were all wrong. Troubleshooting in progress.
ISA1 (M229) and ISA2 (M231) clones correct but contained messed-up suffixes. Other clones which, by digestion, seem to have intact XbaI and Spe1 sites have been submitted for sequencing.

Plac-dxs-lims-appY device is ready to test. Hope to do next week if the GC is available.
Attempts to make crt construct failed for some unexplained reason - apparently no DNA in the ligations.

Submitted for sequencing M229 (ZmISA1), 231 (ZmISA2), 233 (cex mutant), 237 (cenA), 243 (rbs+glgC), 245 (rbs+glgC16), 240 and 247 (bglX).
Digests of M243 and M245 to add lac promoter J33207. Ligations L78, L79?
Ligation L80? to add PcstA (M130) to xylE reporter gene (J33204).
Also found one blue colony on plate 203 (PcstA+lacZ’) which I missed before, so subbed this to a fresh plate (plate 226). What were plates 224 and 225? Probably subcultures from 218-221 and 222-223 (rbs+glgC/C16 and crtBI+dxscrtE).

Minipreps M248-M253 of possible crtBI-dxscrtE clones. Gels 211, 212 show that these all seem to be dxs+crtE without crtBI.

Sequencing results. M229 is ZmISA1 but suffix is messed up, SpeI site not intact. Likewise M231 for ZmISA2. M233 is cex mutant, seems fine. M237 is cenA, fine. M243 and M245 are rbs+glgC/C16. M240 and 247 are not bglX.
Nice blue colonies for Plac+glgC/C16 (J33207 includes lacZ’ as tag to make selecting correct colonies easier).
Digested M233 (cex mutant) and M237 (cenA) to add rbs. Ligations L81, L82.
Digested X13 (crtBI) and X21 (dxscrtE) to add J33207 Plac_lacZ’ tag to both for easier selection of recombinants. Excised from gel 212 (oops, two gels both numbered 212 – call this 212B). Ligations L83, L84.
Digested M230, 231, 232 (ZmISA2) to check for intact SpeI site. M231 is not intact, as indicated by sequencing, but M230 and M232 look OK.

Transformed L83 (Plac+crtBI) and L84 (Plac+dxscrtE), plates 235-238.
Minipreps 254, 255 (Plac+rbs+glgC), 256, 257 (Plac+rbs+glgC16), 258 (PcstA+lacZ’), 259 (PcstA+xylE). Gel 213, all look OK.

Submitted for sequencing M254, 256, 258, 259.
Minipreps M260-265 of more ZmISA1 patches. PstI digests, only M265 looks good (gel 214).
Overnight cultures of Plac+glgC/C16 with and without 40 mM glucose to test for glycogen, also limonene generator as control. IPTG in all. Also PcstA-lacZ’ and PcstA-xylE with and without 40 mM glucose to test repression of this promoter.

PCR P101, P102 to add rbs to cex and cenA (template = L81, L82). Look OK on gel 215.
Digest M265 (ZmISA1) to check SpeI site intact – looks OK (also gel 215).
Minipreps M266-269 (last 4 ZmISA1 patches). None look good, but M269 might be OK (also gel 215).
PcstA-xylE assays good : see spreadsheet.
Glycogen assays look good: see Powerpoint file.
Analytical PCR with L74 as template to diagnose problem with bglX ligation (not numbered as will not be used for cloning).

Gel 216 of bglX diagnostic PCR. All combinations of vector and insert-based primers give products indicating that ligations is occurring at both ends, so that’s not the problem.
Digests of possible ZmISA1 clones M229, M265 and M269 show that M229 lacks SpeI site, M265 has it, and M269 looks wrong.
Cloning P101, P102 (rbs+cex, rbs+cenA) in Edinbrick1: ligations L85, L86.
Might bglX give blue colonies? Try cloning in Edinbrick2 instead: ligation L87.