Part:BBa_K4162118

ribozyme+RBS+CDS module: crtEBIY

Introduction

This biobrick was created through overlapping PCR of BBa_K4162009(ribozyme+B0_RBS+crtE), BBa_K4162013(ribozyme+T7_RBS+crtB), BBa_K4162016(ribozyme+T7_RBS+crtI) and BBa_K4162019(ribozyme+T7_RBS+crtY). These genes are a part of the carotenoid biosynthesis pathway and together, this biobrick converts farnesyl pyrophosphate to beta-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 nullified and each cistron can initiate translation with comparable efficiency.

We transfected this biobrick into E. coli to build single-cell factory for beta-carotene production. Coding sequences of crtEBIY are separated by ribozyme sequences. In this part, the RBS of crtBIY has equal intensity while the RBS of crtE is significantly stronger than the others. Since crtE catalyzes the first step of the carotenoid reaction chain, the concentration of substrate for the reaction catalyzed by this enzyme is significantly higher than for the next three steps of the reaction. To avoid more serious flux imbalance problems, we boosted the RBS intensity of crtE only in this biobrick and explored whether the carotenoid production of the strain could be significantly enhanced.

Characterization

定性1标题

定性1文字

File:T--Fudan--定性1图片不要用中文做文件名.png

Figure 1. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性1小结

定性2标题

定性2文字

File:T--Fudan--定性2图片不要用中文做文件名.png

Figure 2. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性2小结

定性3标题

定性3文字 ^[1]. 又一个参考文献在一对尖括号内，例子里的参考文献格式是pubmed直接copy的

File:T--Fudan--定性3图片不要用中文做文件名.png

Figure 3. 图标题. 图注引用的不要忘了写某某人某某年的出处

定性3小结

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 2202
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1681
Illegal NgoMIV site found at 1811
Illegal AgeI site found at 839
1000
COMPATIBLE WITH RFC[1000]

↑ T7 phage factor required for managing RpoS in Escherichia coli. Tabib-Salazar A, Liu B, Barker D, Burchell L, Qimron U, Matthews SJ, Wigneshweraraj S. Proc Natl Acad Sci U S A, 2018 Jun 5;115(23):E5353-E5362. PMID:29789383

[1] T7 phage factor required for managing RpoS in Escherichia coli. Tabib-Salazar A, Liu B, Barker D, Burchell L, Qimron U, Matthews SJ, Wigneshweraraj S. Proc Natl Acad Sci U S A, 2018 Jun 5;115(23):E5353-E5362. PMID:29789383

[1]