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

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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 assemblyUpstream partDownstream partBackbone
A1’RBSaiiApSB2K3
A2’RBSdspBpSB2K3
A3’RBSdspB+HISpSB2K3
A4’RBSaiiA+HISpSB2K3
 
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 assemblyUpstream partDownstream partBackbone
A1”RBS + aiiADouble terminatorpSB1A3
A2”pLASRBS + dspBpSB1A3
A3”pLASRBS + dspB+HISpSB1A3
A4”RBS + aiiA+HISDouble terminatorpSB1A3
 
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 assemblyUpstream partDownstream partBackbone
A1”’pLAS+RBS+dspBRBS+aiiA+DtermpSB1C3
A2”’pLAS+RBS+dspB+HIS>RBS+aiiA+HIS+DtermpSB1C3
 
Then the whole transformation protocol occurred and the correct clones were confirmed with sequencing.
 
 
 
 

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