Designed by: Weiwen Chen Group: iGEM22_Fudan (2022-10-11)
ribozyme+RBS+CDS module: crtYBEI
This biobrick was created through overlapping PCR of Part:BBa_K4162020 (ribozyme+J6_RBS+crtY), Part:BBa_K4162013 (ribozyme+T7_RBS+crtB), Part:BBa_K4162010 (ribozyme+T7_RBS+crtE) and Part:BBa_K4162016 (ribozyme+T7_RBS+crtI). 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 and each cistron can initiate translation with comparable efficiency.
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-crtY 5-ATGCAACCGCATTATGATCTGATTC-3; > rev320crtB 5-CCTTCCAGATGATCAAACGCGTAAG-3; > rev320crtE 5-ATGAGAATGAATGGTAGGGCGTC-3; > rev320crtI 5-GGATTAAACTGCTGAATCTGCGCTTC-3; > rev320crtY 5-CCGCGGTATCCATCCACAAG-3.
Figure 2. The centrifuge tubes containing module crtYEBI BBa_K4162117 (first from the left) and module crtYBEI (second from the left) contain visible yellow bacterial pellet.
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 3425
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1911 Illegal NgoMIV site found at 2041 Illegal AgeI site found at 3076
