Part:BBa_K4162101

ribozyme+RBS+CDS module: crtIY

Introduction

This biobrick was created through overlapping assembly of Part:BBa_K4162016 (ribozyme+RBS+crtI) and Part:BBa_K4162019 (ribozyme+RBS+crtY). These genes are a part of the carotenoid biosynthesis pathway.

Worth to Notice

Coding sequences of crtI and Y are under T7 RBS Part:BBa_K4162006.

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-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. A、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: pET28 plasmids encoding crtE+crtI without any tag were transformed into BL21(DE3) HI-Control strain, single clones (EI4, EI5) were picked for liquid LB culture. Lane 6~17: pET28 plasmids encoding crtE+crtI, crtI+crtY, crtB+crtE without any tag were transformed into BL21(DE3) Hi-Control strain, single clones (EI8, IY10, BE11, BE12, BE13, BE14) 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.

Figure 3. 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+crtEwithout 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.

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 152
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]