Part:BBa_K4162005

Hammerhead ribozyme

Introduction

Hammerhead ribozyme was first found in the genome of viruses and viroids. It involved in the processing of RNA transcripts based on rolling-circle replication. The tandem copy of RNA sequence will be generated in the roll ring replication, and the self-cleaving activity of ribozyme can ensure the generation of RNA copy of unit length.

The secondary structure of hammerhead ribozyme resembles a hammer. According to different open helix tips, hammerhead ribozymes can be divided into three types: TypeⅠ, Type II and Type III. The catalytic center of ribozyme consists of 15 highly conserved bases surrounded by three helixs(HelixⅠ, Helix II and Helix III). The long-range interaction between HelixⅠ and Helix II can help stabilize the conformation of the catalytic center of the enzyme and improve the catalytic efficiency (Figure 1). While working, ribozymes utilize a network of defined hydrogen bonds, ionic and hydrophobic interactions to generate catalytic pockets, which capitalize on steric constraints to generate in-line cleavage alignments and general acid-base chemistry to catalyze site-specific cleavage of the phosphodiester backbone.

Figure 1. Overall tertiary structure of different types of hammerhead ribozymes.Blue marks highly conserved bases. The black arrow marks the digestion site. Red marks the long-range interaction between HelixⅠand Helix II.(Jimenez, Randi M et al. 2015)

Usage and Biology

We construct a ribozyme-assisted polycistronic co-expression system (pRAP) by inserting ribozyme sequences between CDSs in a polycistron. 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. In our project, we used ribozyme to build our crtEBIY biobrick.

Characterization

Successful protein expression

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. The tail of the coding sequence (CDS) can interfere with the head of the ribosome binding site (RBS), which can hinder RBS from combining to ribosomes. Such shortage occurred when we assembled crtEBIY sequentially only to find incomplete expressions of our target proteins.

We first assemble four basic modules: BBa_K4162010(ribozyme + T7_RBS + crtE), BBa_K4162013(ribozyme + T7_RBS + crtB), BBa_K4162016(ribozyme + T7_RBS + crtI) and BBa_K4162019(ribozyme + T7_RBS + crtY). Next, we apply overlapping PCR to assemble composite modules which contain 2-3 random basic modules. Finally, we obtained several biobricks composed of four basic modules assembled in different orders. SDS-PAGE showed that the proteins in each module were successfully expressed and were intact proteins rather than short peptides after incomplete expression.

Figure 1. 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, 6~9, 11~17: pET28 plasmids encoding crtYB, IB, EY separated by self-cleaving ribozyme, crtB, crtE, crtY without any tag were transformed into BL21(DE3) Rosetta strain, single clones(1YB1/IB7/IB8/1B1/EY3/EY4/1E1/1Y1)were picked for liquid LB culture. Lane 10: pET28 plasmids encoding crtI were transformed into BL21(DE3) Hi-control Rosetta strain, single clones(1I7)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 2. 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) Rosetta 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) Rosetta 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 Rosetta 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.

Stable expression level

Figure 3. 96-well plate of module crtYEBI(BBa_K4162117). Except for the blank tube marked in black, all tubes had similar beta-carotene content in the bacterial sap.

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

[[|400px|thumb|none|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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]