Difference between revisions of "Part:BBa K2404013:Design"
| Line 12: | Line 12: | ||
> Restriction digest at 37C | > Restriction digest at 37C | ||
| − | - [https://www.neb.com/products/r0535-bsai BsaI] | + | - [https://www.neb.com/products/r0535-bsai BsaI] <br> |
- Enzyme buffer | - Enzyme buffer | ||
- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10003 35S-OTMV] | - Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10003 35S-OTMV] | ||
Revision as of 14:18, 27 October 2017
Luc+ gene under control of the 35S CaMV promoter
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 13
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 13
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 13
Illegal BglII site found at 400
Illegal BglII site found at 1146
Illegal BglII site found at 2270
Illegal BamHI site found at 2116
Illegal XhoI site found at 1350 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 13
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 13
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1442
Design Notes
Experimental design for composite part construction:
We conducted a Type IIS restriction enzyme digest using the BsaI enzyme. The reaction mix includes three level 0 plasmid
> Restriction digest at 37C
- BsaI
- Enzyme buffer
- Level 0 plasmid containing 35S-OTMV
- Level 0 plasmid containing NosT
- Level 0 plasmid containing LUC+
- Level 1 plasmid pGB-A1
> Inactivate enzyme at 80C
> Add T4 ligase
> Transform E.coli and plate onto kanamycin (50ug/ul) plates
This part was created using golden gate assembly, and thus has typeIIS restriction enzyme recognition sites flanking it.
Source
This part was created from various other parts, native to various organisms. The promoter is from cauliflower mosaic virus. The CDS is an adjusted version of the firefly luciferase gene. The terminator is of the nopaline synthase gene from Agrobacterium tumefaciens