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Team:DTU-Denmark/Labwork notebook/Damian
From 2011.igem.org
(Created page with "Project: Creating ΔchiP, ΔchiX, ΔchiXR, ΔlacZ strain Strain used: W3110 Sex: F- Chromosomal Markers: &lambda-, IN(rrnD-rrnE)1, rph-1 Plasmids: pSLD18, pFHC3220 Short desc...") |
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| - | Project: Creating ΔchiP, ΔchiX, ΔchiXR, ΔlacZ strain | + | Project: '''Creating ΔchiP, ΔchiX, ΔchiXR, ΔlacZ strain''' |
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Strain used: W3110 | Strain used: W3110 | ||
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Sex: F- Chromosomal Markers: &lambda-, IN(rrnD-rrnE)1, rph-1 | Sex: F- Chromosomal Markers: &lambda-, IN(rrnD-rrnE)1, rph-1 | ||
Plasmids: pSLD18, pFHC3220 | Plasmids: pSLD18, pFHC3220 | ||
| - | Short description | + | Short description |
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To create a strain with 4 gene-knockouts we decided to use Red swap recombineering system. In order for the recombination to work, two plasmids were required: pSLD18, pFHC3220. The first one….. and the second one…… Then, a 3-day cycle of gene knock-out experiments was repeated four times to delete chip, chiX, chiXR and lacZ. | To create a strain with 4 gene-knockouts we decided to use Red swap recombineering system. In order for the recombination to work, two plasmids were required: pSLD18, pFHC3220. The first one….. and the second one…… Then, a 3-day cycle of gene knock-out experiments was repeated four times to delete chip, chiX, chiXR and lacZ. | ||
| - | Detailed description | + | Detailed description |
Week 1. | Week 1. | ||
Latest revision as of 07:30, 7 July 2011
Project: Creating ΔchiP, ΔchiX, ΔchiXR, ΔlacZ strain
Strain used: W3110
Sex: F- Chromosomal Markers: &lambda-, IN(rrnD-rrnE)1, rph-1
Plasmids: pSLD18, pFHC3220
Short description
To create a strain with 4 gene-knockouts we decided to use Red swap recombineering system. In order for the recombination to work, two plasmids were required: pSLD18, pFHC3220. The first one….. and the second one…… Then, a 3-day cycle of gene knock-out experiments was repeated four times to delete chip, chiX, chiXR and lacZ.
Detailed description
Week 1. PCR of DNA containing kanR and homologous regions to regions encompassing our genes of interest. Transforming E. coli with pFHC3220; Transforming E. coli with linear pieces of DNA and selecting for gene deletions - ΔchiP, ΔchiX, ΔchiXR, ΔlacZ;
Week 2. Growth of E. coli in arabinose minimal medium containing amp and ery. Selecting for loss of kan resistance after first round of knock-out
