Designed by: Weiwen Chen Group: iGEM22_Fudan (2022-10-05)
ribozyme+RBS+CDS module: crtIYEB
Introduction
This biobrick was created through standard biobrick assembly of Part:BBa_K4162016 (ribozyme+RBS+crtI), Part:BBa_K4162019 (ribozyme+RBS+crtY), Part:BBa_K4162010 (ribozyme+RBS+crtE) and Part:BBa_K4162013 (ribozyme+RBS+crtB). These genes are a part of the carotenoid biosynthesis pathway and together, this biobrick converts farnesyl pyrophosphate to β-carotene. In this part, crtIYEB are separated by hammerhead ribozyme, which can perform self-cleaving. Therefore, the long piece of mRNA it transcribes into will be spontaneously separated into four segments, which correspond to crtI, Y, E and B repectively. The translation process of these four enzymes is independent of each other, and the proportion of the expression products of four enzymes can be accurately adjusted by monitoring the intensity of RBS.
However, due the nature of proteins, we could not seen yellow bacteria pellet, nor confirming the expression of crtE by SDS-PAGE. These facts motivated us to build Part:BBa_K4162117 and Part:BBa_K4162118. Combining the power of ribozyme-assisted mRNA self-cleavage and different RBS driven gene expression, we successfully produce &beta-carotene, and later retinol. For more details, please visit the later two parts page and our proof of concept page.
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-crtI 5-ATGAAACCAACTACGGTAATTGGTGC-3; > rev320crtB 5-CCTTCCAGATGATCAAACGCGTAAG-3; > rev320crtE 5-ATGAGAATGAATGGTAGGGCGTC-3; > rev320crtI 5-GGATTAAACTGCTGAATCTGCGCTTC-3; > rev320crtY 5-CCGCGGTATCCATCCACAAG-3.
SDS-PAGE
Figure 2. SDS-PAGE. D、IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 1~4、6～15: pET28 plasmids encoding crtIYBE separated by self-cleaving ribozyme，crtI+crtY+crtB+crtE，crtI+crtY，crtE+crtI，crtB+crtE without any tag were transformed into BL21(DE3) HI-Control strain, single clones (6a, 4a, IY10, EI8，BE11，BE12，BE13) were picked for liquid LB culture. 6=crtIYEB，4=crtIYBE. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analysed by SDS-PAGE (14% separation gel). Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.
Figure 3. SDS-PAGE. IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 4~16: pET28 plasmids encoding crtBEI separated by self-cleaving ribozyme, crtI, crtEI, crtB, crtEB, crtIYEB separated by self-cleaving ribozyme without any tag were transformed into BL21(DE3) HI-Control strain, single clones (BEI/1I7/1EI/1B1/EB5/EB6/IYEBa) were picked for liquid LB culture. Lane 1~2: pET28 plasmids encoding BCMO-ybbO-tdMCP-EGFP were transformed into BL21(DE3) HI-Control strain, single clones (2BY14tG14) were picked for liquid LB culture. Lane 3~4: pET28 plasmids encoding BCMO-ybbO were transformed into BL21(DE3) Hi-control strain, single clones (BY14hi) were picked for liquid LB culture. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analyzed by SDS-PAGE (4~20% gradient gel, Tanon brand).Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.
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 197
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 4479 Illegal NgoMIV site found at 4609 Illegal AgeI site found at 3637
