Team:Cambridge/Lab book/Week 13


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Week: 3 4 5 6 7 8 9 10 11 12 13 14


Monday (17/09/12)

Ratiometrica - Lux

Testing of the 2.0 construct:

Numbers 33,35,36 have correct sequence as of being sent to us. However they were not visibly luminescent. *34 was.

Overnight cultures of each of the 6 culture lines sent were set up. Additionally, inducing plates were streaked, including the 2010 lux biobrick as a luminescence control and pjs130 as an iptg induction control.

Primers ordered to ligate 2.0 construct into pjs130 for insertion into bacillus.

Tuesday (18/09/12)

Ratiometrica - Lux

None of restreaked plates visibly luminescent, aside from *34 (faint), even under photon counting camera. Pjs130 not green, so iptg induction potentially didn't work. LuxBB induced and luminescent.

Cultures induced with 1mM iptg or 3mM arabinose for luxBB, imaged after 3 hrs. Some extremely faint luminescence for 33,34,38 on photon counting camera.

Cultures miniprepped and restriction digests carried out w/spa1. Results are shown. Results indicate problem with the miniprep and/or damage to the construct - expected bands at 6625, 2838, 1876 and 53 bp.

Digests are of 33-38, each miniprep, cut and uncut, between 2 ladders.

Wednesday (19/09/12)

Ratiometrica - Lux

Digest of 2.0 miniprep with different enzyme, hindIII, carried out. 15ul template DNA used in a 20ul reaction. Again, results were odd. Cut and uncut minipreps ran.
HindIII digest

Cultures of ratiometrica (fluorescent) were miniprepped to be transformed into bacillus. A restriction digest was performed on these minipreps, and produced bands of the correct size.

Ratiometrica Digest
Ratiometrica Digest Prediction

Thursday (20/09/12)

Transformations of XL1-blue cells with resuspended construct plasmid from DNA 2.0 yielded orange colonies, with some white, and some showing sectoring, indicating insert loss.

An image showing the variation between fresh transformants. Note sectoring of individual colonies, indicating that the insert is being lost as the colonies grow.

Plates all appear to be luminescent. A random selection of colonies was picked and patched in order to investigate whether luminescence cosegregated with orange colour.

Friday (21/09/12)

Initial pick and patch experiments showed cosegregation, but we did not have immediate access to a photon-counting camera or a DSLR capable of long exposures, so we did not obtain images. Representative colonies were streaked out onto a fresh plate to be imaged later.

Saturday (22/09/12)

Sunday (23/09/12)