Difference between revisions of "Part:BBa K4162028"
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| − | Gene ybbO tagged with tdMCP can be combined with the stem ring structure of MS2, so that retinol dehydrogenase expressed by gene ybbO can be enriched in the area of TEARS. This part can be used to assemble polycistrons in | + | ===Introduction=== |
| + | [[File:T--Fudan--logo-Rester-rectangle.jpg|100px|right|2022 Fudan]] | ||
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| + | Gene ''ybbO'' tagged with tdMCP can be combined with the stem ring structure of MS2, so that retinol dehydrogenase expressed by gene ''ybbO'' can be enriched in the area of [https://www.biorxiv.org/content/10.1101/2020.07.02.182527v2 TEARS]. This part can be used to assemble polycistrons in ''E. coli''. | ||
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| + | __TOC__ | ||
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| + | ===Characterization=== | ||
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| + | ====SDS-PAGE==== | ||
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| + | [[File:T--Fudan--protein--2YtG15,2BY14tG14,YEB,Y+E+B.png|400px|thumb|none|'''Figure 1. 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 10~17: pET28 plasmids encoding crtYEB separated by self-cleaving ribozyme, crtY, crtE, crtB without any tag were transformed into BL21(DE3) HI-Control strain, single clones(YEBd, YEBe, 1Y1, 1E1, 1B1)were picked for liquid LB culture. Lane 1~2, 4~8: pET28 plasmids encoding ybbO-tdMCP-EGFP, BCMO-ybbO-tdMCP-EGFP were transformed into BL21(DE3) HI-Control strain, single clones(2YtG15, 2BY14tG14)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. Purple arrows point to ybbO protein.]] | ||
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Latest revision as of 11:40, 12 October 2022
ribozyme + T7_RBS + ybbO-tdMCP-EGFP
Introduction
Gene ybbO tagged with tdMCP can be combined with the stem ring structure of MS2, so that retinol dehydrogenase expressed by gene ybbO can be enriched in the area of TEARS. This part can be used to assemble polycistrons in E. coli.
Contents
Characterization
SDS-PAGE
Figure 1. 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 10~17: pET28 plasmids encoding crtYEB separated by self-cleaving ribozyme, crtY, crtE, crtB without any tag were transformed into BL21(DE3) HI-Control strain, single clones(YEBd, YEBe, 1Y1, 1E1, 1B1)were picked for liquid LB culture. Lane 1~2, 4~8: pET28 plasmids encoding ybbO-tdMCP-EGFP, BCMO-ybbO-tdMCP-EGFP were transformed into BL21(DE3) HI-Control strain, single clones(2YtG15, 2BY14tG14)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. Purple arrows point to ybbO protein.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1612
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1612
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1612
Illegal AgeI site found at 109
Illegal AgeI site found at 702 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1192
Illegal BsaI site found at 1546