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Team:Groningen/Template/MODULE/Notebook/secretion/week9
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| - | < | + | Obtaining the anti-<i>Pseudomonas aeruginosa</i> system with Three-A assembly |
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| - | Extra amounts of <i>dspB</i>, | + | Extra amounts of <i>dspB</i>, ssUSP45DspB with His-tag, <i>aiiA</i>, ssUSP45aiiA with His-tag, <i>pLAS</i>, the RBS, the double terminator, pSB2k3 vector, pSB1C3 vector and pSB1A3 vector were miniprepped to be used for the assembly |
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| - | + | <i>aiiA</i>, <i>dspB</i>, ssUSP45DspB with His-tag and ssUSP45aiiA with His-tag were digested with XbaI and PstI, and the RBS was cut with EcoRI and SpeI | |
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| + | After transformation, the plasmids were isolated, and confirmed by sequencing | ||
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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
