Part:BBa_K4162118

ribozyme+RBS+CDS module: crtEBIY

Introduction

This biobrick was created through overlapping PCR of BBa_K4162009(ribozyme+B0_RBS+crtE), BBa_K4162013(ribozyme+T7_RBS+crtB), BBa_K4162016(ribozyme+T7_RBS+crtI) and BBa_K4162019(ribozyme+T7_RBS+crtY). These genes are a part of the carotenoid biosynthesis pathway and together, this biobrick converts farnesyl pyrophosphate to β-carotene. In this part, the RNA sequences of hammerhead ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo. Self-interaction of the polycistron can be avoid. Ribozyme-assisted polycistron expression ensures each cistron can initiate translation with comparable efficiency.

Usage and Biology

We transfected this biobrick into E. coli to build single-cell factory for β-carotene production. Coding sequences of crtEBIY are separated by ribozyme sequences. In this part, the RBS of crtBIY has equal intensity while the RBS of crtE is significantly stronger than the others. Since crtE catalyzes the first step of the carotenoid reaction chain, increase the concentration of product catalyzed by this enzyme is beneficial for the remaining three steps. To avoid more serious flux imbalance problems, we boosted the RBS intensity of crtE only in this biobrick and explored whether the carotenoid production of the strain could be significantly enhanced.

Comparing to BBa_K4162021, we use B0_RBS rather T7_RBS to drive the translation crtY. B0_RBS is stronger than T7_RBS, which means greater chance for ribosomes to start the translation. Please note that the expression of crtE is obscure for BBa_K4162021 under IPTG induction, which also motivated us to enhance the expression of crtE.

Characterization

Agarose gel electrophoresis

Figure 1. 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.

Successful production of β-carotene

Figures 2 show that E. coli transfected with plasmid expressing this biobrick successfully produce β-carotene.

Figure 2. Overnight culture of bacterial expressing module crtEBIY was centrifuged at 13000 rpm for 1 minute.

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 2202
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1681
  Illegal NgoMIV site found at 1811
  Illegal AgeI site found at 839
  
• 1000
  COMPATIBLE WITH RFC[1000]