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

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Revision as of 21:09, 17 October 2014

September 29 - October 5

goal: Obtaining pLASs with 3-a assembly

Extra amounts of dspB, dspB+HIS, aiiA, aiiA+HIS,aiiA+HIS, pLAS, RBS, double terminator, pSB2k3, pSB1C3 and pSB1A3 were miniprepped to be used for the assembly

1 ug of aiiA, dspB, dspB+HIS and aiiA+HIS with XbaI and PstI and 1 ug of 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

Then the whole transformation protocol occurred and the correct clones were confirmed with sequencing.

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