Designed by: Weiwen Chen Group: iGEM22_Fudan (2022-10-07)
ribozyme+RBS+CDS module: crtYEBI
Introduction
This biobrick was created through overlapping PCR of BBa_K4162020(ribozyme+J6_RBS+crtY), BBa_K4162010(ribozyme+T7_RBS+crtE), BBa_K4162013(ribozyme+T7_RBS+crtB) and 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. Thus, self-interaction of the polycistron can be avoid, and each individual cistron containing RBS+CDS can initiate translation with comparable efficiency.
Comparing to BBa_K4162021, we use J6_RBS rather T7_RBS to drive the translation crtY. J6_RBS is weaker than T7_RBS, which means less chance for ribosomes to start the translation. Reduced expression of crtY may help the expression of crtE, which is obscure for BBa_K4162021 even under IPTG induction.
We transfected this biobrick into E. coli to build single-cell factory for β-carotene production. Coding sequences of crtYEBI are separated by ribozyme sequences. In this part, the RBS of crtEBI has equal intensity while the RBS of crtY is significantly weaker than the others. Since crtY catalyzes the last step of the carotenoid reaction chain, we speculate that the enzyme catalyzes a significantly lower concentration of substrate than the first three steps of the reaction. To avoid the problem of flux imbalance in biosynthesis as well as to reduce unnecessary metabolic stress on cells, we intentionally weakened the RBS intensity of crtY.
Our unsuccessful biobrick BBa_K4162021 supports our guess as well.
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-crtY 5-ATGCAACCGCATTATGATCTGATTC-3; > rev320crtB 5-CCTTCCAGATGATCAAACGCGTAAG-3; > rev320crtE 5-ATGAGAATGAATGGTAGGGCGTC-3; > rev320crtI 5-GGATTAAACTGCTGAATCTGCGCTTC-3; > rev320crtY 5-CCGCGGTATCCATCCACAAG-3.
Successful protein expression
Figure 2. 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 1~2: pET28 plasmids encoding crtEBIY without any tag were transformed into BL21(DE3) HI-Control strain, single clones (6b) were picked for liquid LB culture. Lane 3~12: pET28 plasmids encoding crtYEBI without any tag were transformed into BL21(DE3) Hi-Control strain, single clones (Hi-7, Hi-7a, Hi-7b, Hi-7c, Hi-7d) were picked for liquid LB culture. Lane 14~17: pET28 plasmids encoding crtB, crtE, crtI, crtYwithout any tag were transformed into BL21(DE3) HI-Control strain, single clones (1B1, E1, I7, Y1) 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.
Produce β-carotene
Figures 2 to 4 show that E. coli transfected with this biobrick successfully expressed the target enzyme and yielded β-carotene. In Figure 4, it can be seen that module YEBI corresponds to a darker orange color of the post-centrifugation precipitation compared to module YBEI(BBa_K4162119), characterizing the superior carotenoid yielding ability of module YEBI.
Figure 3. 96-well plate of module crtYEBI. Except for the blank control well marked in black, all clones growing different wells had similar β-carotene content in the bacterial pellet.
Figure 4. The centrifuge tube containing a visible yellow precipitation on the right is the module crtYEBI. The bacterial pellet was proceeded for miniprep. After P1→P2→P3, 10-minute centrifugation and transfering the DNA containing supernatant into DNA binding column, we noticed what left, usually white cloudy precipitation, was yellow! Later, when we prepared samples for HPLC from bacterial pellet, we show that these yellow stuff can be extracted into acetone.
Figure 5. The centrifuge tubes containing module crtYEBI (first from the left) and module crtYBEI BBa_K4162119 (second from the left) contain visible yellow bacterial pellet.
HPLC validation
We used <a href="https://mp.weixin.qq.com/s/81zY_LoMratSEOOy7JUaFw" target="_blank">a previous reported</a> protocol for HPLC: Agilent liquid chromatograph (HPLC-DAD); column C18 (250mm); column temperature 30°C; mobile phase methanol:water = 96:4; flow rate 0.8ml/min; detection wavelength 325 nm and 454 nm.
Figure 6. Absorption peak of β-carotene (Sigma cat# C4582) standard solution. Methanol saturated solution of β-carotene, 1:10 diluted in methanol before loading into the column. Blue lines are from the detectors: the upper plane was from the 325-nm detector, and the lower from the 454-nm detector. Pink lines are only for analysis.
Figure 7. Absorption peak of acetone extraction from bacterial DH5α expressing this biobrick. The slightly delayed peaks of β-carotene from E. coli DH5α expressing BBa_K4162117 likely due to acetone used in the extraction. Absorption peaks in Figure 7 is very similar to what in Figure 6, confirming successful production of β-carotene in our engineered bacterial factory.
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 2904 Illegal NgoMIV site found at 3034 Illegal AgeI site found at 2062
