Difference between revisions of "Part:BBa K2429056"
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This part consists of the lac operon present in E.coli bacteria, and is essential for the metabolism of lactose in bacteria. We used it down stream of a hU6 promoter to form a backbone vector for our guide sequences. We used golden gate reactions to insert the desired guide sequences directly in the middle of the LacZ gene. Following transformations, this allowed us to further select which bacterial colonies contained the plasmids with the guide sequences. In the presence of X-gal, colonies with the LacZ gene still intact would appear blue, and wouldn't have plasmids with guide sequences in them. Colonies that appeared white would had disruption of the LacZ gene, and was likely to contain the desired plasmid. | This part consists of the lac operon present in E.coli bacteria, and is essential for the metabolism of lactose in bacteria. We used it down stream of a hU6 promoter to form a backbone vector for our guide sequences. We used golden gate reactions to insert the desired guide sequences directly in the middle of the LacZ gene. Following transformations, this allowed us to further select which bacterial colonies contained the plasmids with the guide sequences. In the presence of X-gal, colonies with the LacZ gene still intact would appear blue, and wouldn't have plasmids with guide sequences in them. Colonies that appeared white would had disruption of the LacZ gene, and was likely to contain the desired plasmid. | ||
Latest revision as of 23:35, 25 October 2017
LacZ Casette
This part consists of the lac operon present in E.coli bacteria, and is essential for the metabolism of lactose in bacteria. We used it down stream of a hU6 promoter to form a backbone vector for our guide sequences. We used golden gate reactions to insert the desired guide sequences directly in the middle of the LacZ gene. Following transformations, this allowed us to further select which bacterial colonies contained the plasmids with the guide sequences. In the presence of X-gal, colonies with the LacZ gene still intact would appear blue, and wouldn't have plasmids with guide sequences in them. Colonies that appeared white would had disruption of the LacZ gene, and was likely to contain the desired plasmid.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 349
Illegal XbaI site found at 376
Illegal PstI site found at 388 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 349
Illegal PstI site found at 388 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 349
Illegal BamHI site found at 370 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 349
Illegal XbaI site found at 376
Illegal PstI site found at 388 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 349
Illegal XbaI site found at 376
Illegal PstI site found at 388 - 1000COMPATIBLE WITH RFC[1000]