Part:BBa_K4720105

Luciferase gene cassette

This is a theoretical part that is a luxCDABE operon that can be used as reporter. The expression of this gene cassette should result in luciferase bio luminescence. The Lux cassette was obtained from the plasmid pAKgfplux1 from AddGene [1]. We planned on performing a site directed mutagenesis to remove a XbaI restriction site to make the part RFC[10] compatible.

These are the Primers for the mutagenesis (5' to 3'):

MutLux_Fwd: CATTAATGAATTGCCGAATAACCTAGATTTTGAAGGCCATAAATTGGGTGCTGAAGTC

MutLux_Rev: GGCCTTCAAAATCTAGGTTATTCGGCAATTCATTAATGGGTAGACTGAGATAATCAAACCC

The PCR fragment can be circularized by Gibson Assembly with (38 bp overlapping)

In order to move the lux cassette into a iGEM vector with prefix and suffix, we designed the following primers for the insertion into Gibson Assembly (40 bp overlapping) into the pSB1A3 vector:

Lux_Fwd: cttgcccttttttgccggactgcagcggccgctactagtaTCAACTATCAAACGCTTCGGTTAAGC

Lux_Rev: ttcgctaaggatgatttctggaattcgcggccgcttctagATGACTAAAAAAATTTCATTCATTATTAACGGCCAGG

The pSB1A3 backbone can be amplified with the following primers:

pSB1A3_Fwd: TACTAGTAGCGGCCGCTGCAG

pSB1A3_Rev: TCTAGAAGCGGCCGCGAATTCC

Due to time constraints the construct was not obtained. We leave the sequence here, with information on how to obtain it, for future iGEM teams.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5274
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2931
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1311
  Illegal SapI site found at 3450