Lab Oct 15 2009

5.30am

There was an absolute lack of growth in the K235032 + IPTG tube and I think I may have forgotten to swirl a colony. That has now been done that and placed in the 37°C (non-shaking) waterbath along with all the other original broth cultures.

Made five new subcultures for each of K235032 Kan, 33 Kan, 36 Kan, 37 Chl, 05 Chl, J04630 Amp/Kan, and 32 Kan + IPTG. The 'A' broth cultures were used for the first four, and the 'B' broth culture was used for the + IPTG, as the IPTG culture had no growth. 2 µL of LB were used with 30 µL of culture. 2 µL of 1M IPTG were used, for a final concentration of 1mM. No antibiotic was used in any of them.

6am

One set of each K235032 A, 33 A, 36 A, 37 A, 05, J04630, and 32 B + IPTG, were placed in 30, 34, 37 and 42°C (non-shaking) incubation.

Temperature 	Room temperature 	30°C 	34°C 	37°C 	42°C K235032 A expected result 	/ 	/ 	/ 	/ 	/ 	This should be repressed (no green) without lactose or IPTG independent of temperature. K235032 A actual result K235032 B + IPTG expected 	G 	G 	G 	G 	G 	This should be induced by IPTG and produce green independent of temperature. K235032 B +IPTG actual 	 	  	  	G K235033 A expected 	/ 	/ 	/ 	/ 	/ 	Negative control to account for microscope artifacts - there is no colour producing part at all. K235033 A actual K235036 A expected 	/ 	/ 	R 	R 	R 	This should be controlled by the 32°C thermometer and red should be repressed under 32°C. K235036 A actual K235037 A expected 	/ 	/ 	G 	G 	G 	This should be controlled by the 32°C thermometer and green should be repressed under 32°C. K235037 A actual K235005 A expected 	R 	R 	R 	R 	R 	This has no promoter but might produce limited red independent of temperature. K235005 A actual J04630 expected 	G 	G 	G 	G 	G 	This has no promoter but might produce limited green independent of temperature. J04630 actual

6.10am

Minipreps started for 'A' and 'B' of K235032 Kan, 33 Kan, 36 Kan, 37 Chl. The unpaired 05 Chl was also done to balance out the centrifuge, and it will be interesting to see if it's band has changed at all after a new round of transformation.

8.20am

Gel started. Reused the gel from yesterday.

9.30am

Excellent gel results! Reusing the gel caused no problems. 36 B was very faint, but the dye was very faint while running as well and I think I may have loaded it through the gel, allowing it to drift away.

Oct 15 gel 1.tif K235032 A Ladder 	K115020 	E0840 	K235030 Kan 	K235030 Chl 	K235031 Band 	Band 	Band 	Band 	Band 33 A 	36 A 	37 A 	32 B 	33 B 	36 B 	37 B Band 	-Reused- 	Band 	Band 	Band 	Band 	Band 	Faint band 	Band

I will be in at 2.30pm to go do the flourescence microscopy in (I think) Cun 065. Depending on how the time goes today I will be doing assemblies and transformations this afternoon or this evening.

3.30pm

A very frustrating day because the results were looking good and I had to leave! I was able to look at K235032 + IPTG grown at 37oC and it definitely had green flourescence. Then I ran out of time! But I'll be back in tomorrow morning, and I will be doing all of the samples if I can. At this point I definitely feel we have enough to send something in even if they end up being twins, so I will be planning on shipping parts on Monday.

I will be back tonight at 9.30pm to do new assemblies, and I think at some point resubculture the high temp samples.

Thoughts:

Looking at older plates, K235005 started showing red colour after being in the fridge for a while. It is also missing any promoter, but it's interesting to note that tendency to express more colour after being refrigerated for a while, and this has also shown in other parts as well. Is this because they are dying, simply have more time to build up the colour, or something else?

If things are showing on the flourescence microscope, the next step would be to assemble true negative controls consisting of J23102 + B0034 + K235005, and J23102 + B0034 +J04630; namely the same promoter and reporter with a standard RBS instead of the temperature sensitive one.

I'm a little worried that even if these are working it will be hard to tell because of the possibly very low and/or slow expression. I have asked to use the flourescence microscope this afternoon, at 2.30pm giving eight and a half hours, which I'm not sure will be enough... If I get nothing, I will definitely ask to try again tomorrow on Friday.

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