Part:BBa_K4162108

ribozyme+RBS+CDS module: crtEBI

Introduction

This biobrick was created through overlapping assembly of Part:BBa_K4162010 (ribozyme+RBS+crtE), Part:BBa_K4162013 (ribozyme+RBS+crtB) and Part:BBa_K4162016 (ribozyme+RBS+crtI). These genes are a part of the carotenoid biosynthesis pathway.

Worth to Notice

Coding sequences of crtE, B and I are under T7 RBS Part:BBa_K4162006.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures. The first lane was loaded with D2000 DNA ladder whose sizes were marked on the image. We chose Taq DNA polymerase for its low cost and high reliability, and we designed forward and reverse primers for each carotene synthesis enzyme (crt for short). The PCR reaction was composed of 2 μL 10x Taq polymerase buffer, 16 μL H₂O, 0.5 μL Taq polymerase, 0.5 μL dNTP (10 mM each), 0.5 μL forward primer (10 mM), 0.5 μL reverse primer (10 mM), and 1 μL bacterial culture or 1 colony. Using the same forward primer, and different reverse primers, we were able to detect the composition of various crt genes. After PCR, the correct bacterial clones were sent for Sanger sequencing. Once verified, these clones would be used for further experiments. The sequences of primers are: > 5-crtE 5-ATGACGGTCTGCGCAAAAAAAC-3; > rev320crtB 5-CCTTCCAGATGATCAAACGCGTAAG-3; > rev320crtE 5-ATGAGAATGAATGGTAGGGCGTC-3; > rev320crtI 5-GGATTAAACTGCTGAATCTGCGCTTC-3; > rev320crtY 5-CCGCGGTATCCATCCACAAG-3.

Consistent lycopene production among bacterial clones

Figure 2. 96-well plate of bacterial clones expressing this biobrick. Most, except 3, wells, had similar lycopene content in the bacterial pellets.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2159
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1638
  Illegal NgoMIV site found at 1768
  Illegal AgeI site found at 796
  
• 1000
  COMPATIBLE WITH RFC[1000]