Difference between revisions of "Part:BBa K2404013:Design"
Line 13: Line 13:
 
> Restriction digest at 37C
 
> Restriction digest at 37C
 
- [https://www.neb.com/products/r0535-bsai BsaI] <br>
 
- [https://www.neb.com/products/r0535-bsai BsaI] <br>
−
- Enzyme buffer
+
- Enzyme buffer <br>
−
- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10003 35S-OTMV]   
+
 
−
- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10401 NosT]   
+
- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10003 35S-OTMV]  <br>
−
- Level 0 plasmid containing LUC+
+
 
 +
- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10401 NosT]  <br>
 +
 
 +
- Level 0 plasmid containing LUC+ <br>
 +
 
 
- Level 1 plasmid [https://parts.igem.org/Part:BBa_P10503 pGB-A1]
 
- Level 1 plasmid [https://parts.igem.org/Part:BBa_P10503 pGB-A1]
 +
<br>
  
 
> Inactivate enzyme at 80C
 
> Inactivate enzyme at 80C

Revision as of 14:18, 27 October 2017


Luc+ gene under control of the 35S CaMV promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 13
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 13
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 13
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 13
  • 1000
    INCOMPATIBLE 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

References