Team:Groningen/Template/MODULE/Notebook/secretion/week9

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Latest revision as of 00:46, 18 October 2014

September 29 - October 5

Obtaining the anti-Pseudomonas aeruginosa system with Three-A assembly

Extra amounts of dspB, ssUSP45DspB with His-tag, aiiA, ssUSP45aiiA with His-tag, pLAS, the RBS, the double terminator, pSB2k3 vector, pSB1C3 vector and pSB1A3 vector were miniprepped to be used for the assembly

aiiA, dspB, ssUSP45DspB with His-tag and ssUSP45aiiA with His-tag were digested with XbaI and PstI, and the RBS was cut with EcoRI and SpeI

These were eventually ligated for 3 hours into pSB2K3 and transformed into E. coli NEB cells

Name assembly	Upstream part	Downstream part	Backbone
A1’	RBS	aiiA	pSB2K3
A2’	RBS	dspB	pSB2K3
A3’	RBS	dspB+HIS	pSB2K3
A4’	RBS	aiiA+HIS	pSB2K3

After growing them for 1 day, colony PCR was done on the correct clones, these were inoculated for plasmid extraction

Then A1’, A4’ was digested with XbaI and PstI. A2’ and A3’ was cut with EcoRI and SpeI. Then ligated

Name assembly	Upstream part	Downstream part	Backbone
A1”	RBS + aiiA	Double terminator	pSB1A3
A2”	pLAS	RBS + dspB	pSB1A3
A3”	pLAS	RBS + dspB+HIS	pSB1A3
A4”	RBS + aiiA+HIS	Double terminator	pSB1A3

After doing growing analyzing and miniprepping the correct clones, A1” and A3”was cut with EcoRI and SpeI. A2” and A4” was cut with XbaI and PstI and ligated afterwards

Name assembly	Upstream part	Downstream part	Backbone
A1”’	pLAS+RBS+dspB	RBS+aiiA+Dterm	pSB1C3
A2”’	pLAS+RBS+dspB+HIS>	RBS+aiiA+HIS+Dterm	pSB1C3

After transformation, the plasmids were isolated, and confirmed by sequencing

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-After transformation, the plasmids were isolated and confirmed by sequencing
+After transformation, the plasmids were isolated, and confirmed by sequencing
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