SBB09Ntbk-Madhvi Venkatesh

Madhvi Venkatesh 14:54, 2 February 2009 (EST)

Today, I got my project ball and I am making a new flagellar display system. I need to consider several possibilities and figure out what would be best to make and the source. The project description if on this page: new flagellar display system

Madhvi Venkatesh 15:40, 9 February 2009 (EST)

Possible places to find EtpA homolog (from CTEC):

APEC 01:k1
CFT073
EHEC 0157:H7
Shigella flexneri

Madhvi Venkatesh 15:24, 23 February 2009 (EST)

So I rewrote my construction files after I realized that I can't display a protein on the N and C terminal ends of fliD because they are not facing the surface. The new versions of the construction files including Chris's notes on them are linked from the construction files page. We got oligos last Wednesday and I set up PCRs for the N and C terminal parts and wobble PCRs for the linkers. I purified and digested over the weekend. I also set up the SOEing PCR at the end of last week. Today I ligated the parts into pBca1256 and transformed. I will pick colonies tomorrow and send for sequencing on Wednesday. I also purified and digested the SOEing PCr for removing the restriction site in the N terminal fliD part.

Here are gel pics from the PCRs:

Here is the result of the SOEing PCR for the M1000 part {<N.fliD!} with the restriction site removed:

Here are the results of the other PCRs:

Madhvi Venkatesh 21:23, 27 February 2009 (EST)

Lane	Plasmid	Clone	Enzyme 1	Enzyme 2	Expected Bands	Result
1	pBca1256-M10000	1	EcoRI	XhoI	986 + 2113	correct
2	pBca1256-M10000	2	EcoRI	XhoI	986 + 2113	correct
3	pBca1256-M10001	2	EcoRI	XhoI	986 + 2248	correct (not completely digested)
4	pBca1256-M10002	2	EcoRI	XhoI	986 + 1585	No DNA
5	pBca1256-M10002	1B	EcoRI	XhoI	986 + 1585	correct (not completely digested)
6	pBca1256-M10003	1B	EcoRI	XhoI	986 + 1582	correct (mostly undigested)
7	pBca1256-M10003	2B	EcoRI	XhoI	986 + 1582	correct

Based on this, I am sending M10000-1, M10001-2, M10002-1B, M10003-2B for sequencing.