Part:BBa_K2151200

crtEBIY

. This biobrick was created through standard biobrick assembly of K118014(RBS+crtE), K118006(RBS+crtB), K118005(RBS+crtI) and K118013(crtY). These genes are a part of the carotenoid biosynthesis pathway and together, this biobrick converts converts colourless farnesyl pyrophosphate to orange beta-carotene 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 1974
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1510
  Illegal NgoMIV site found at 1640
  Illegal AgeI site found at 725
  
• 1000
  COMPATIBLE WITH RFC[1000]

Fudan 2022 iGEM team

As many researches indicate, the major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter. Instead of assembling CDSs sequentially, we construct a ribozyme-assisted polycistronic co-expression system (pRAP) by inserting ribozyme sequences between crtEBIY. In the pRAP system, 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 nullified and each cistron can initiate translation with comparable efficiency. Besides, we can precisely manage this co-expression system by adjusting the RBS strength of individual mono-cistrons.

Usage and biology

Our improved part is BBa_K4162117. 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). We transfected this biobrick into E. coli to build single-cell factory for beta-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. Because crtY catalyzes the last step of the carotenoid reaction chain, we guess the concentration of substrate catalyzed by this enzyme is significantly lower than for 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.

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 1μL 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) Rosetta 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) Rosetta 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 beta-carotene

Figures 2 to 4 show that E. coli transfected with this biobrick successfully expressed the target enzyme and yielded beta-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 beta-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

Protocol: 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 325nm & 454nm.

Figure 6. Absorption peak of beta-carotene standard solution. 1:10 dilution of methanolic saturated solution of beta-carotene.

Figure 7. Absorption peak of bacterial extract sample. The beta-carotene output of E. coli transfected with the plasmid carrying BBa_K4162117 is shown in the graph as a distinct absorption peak.