Phase 2: Plasmid Construction and Bacterial Transformations
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'''2. Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids''' | '''2. Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids''' | ||
| - | ::* | + | ::*Cut at EcoRI, XbaI, and PstI restriction sites (cutting at XbaI site reduces background) |
| + | ::*pSB1A3 contains amp resistance | ||
| + | ::*Concentration of digested vector and inserts determined. Ligations were set up for between 40:150 to 40ng:300ng ratio vector:insert | ||
| + | <br><br> | ||
| + | '''3. Bacterial transformation''' | ||
| + | ::*Used ''E.coli'' strain RP8611 (null for all MCP receptors, provided by John S. Parkinson, University of Utah) | ||
::*Verified transformation for all fragments and sequenced genes (results positive in all cases). | ::*Verified transformation for all fragments and sequenced genes (results positive in all cases). | ||
<br> | <br> | ||
<br> | <br> | ||
| - | ''' | + | '''4. Significance: |
::*We now have all the fundamental components for the project ahead | ::*We now have all the fundamental components for the project ahead | ||
::*Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes | ::*Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes | ||
::*Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site | ::*Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site | ||
| + | <br><br> | ||
| + | [[Rice University 2006|Back to Rice iGEM]] | ||
Latest revision as of 06:25, 2 November 2006
1. Isolated ptetRBS and pSB1A3 plasmids from BioBricks
- Attached tet promoter with Ribosome Binding Site to GFP gene, transformed XL-1 E. coli and observed positive expression
2. Ligated ComQX/P/A and Tsr fragments from Phase 1 into pSB1A3 plasmids
- Cut at EcoRI, XbaI, and PstI restriction sites (cutting at XbaI site reduces background)
- pSB1A3 contains amp resistance
- Concentration of digested vector and inserts determined. Ligations were set up for between 40:150 to 40ng:300ng ratio vector:insert
3. Bacterial transformation
- Used E.coli strain RP8611 (null for all MCP receptors, provided by John S. Parkinson, University of Utah)
- Verified transformation for all fragments and sequenced genes (results positive in all cases).
4. Significance:
- We now have all the fundamental components for the project ahead
- Can proceed in constructing the hybrid quorumtaxis receptor using ComP and Tsr genes
- Will need to perform quick-change mutagenesis for ComA to remove internal SpeI site
