Part:BBa_K4115044
J23101-B0034-tagRFP-B0015
| J23101-B0034-tagRFP-B0015 | |
|---|---|
| Function | Gene reporter |
| Use in | E. coli |
| RFC standard | RFC 10, RFC 10 compatible |
| Backbone | pUC, pBBR1 |
| Submitted by | ShanghaiTech_China |
This composite part is used to express the tagRFP(BBa_K4115013) by a constitutive promoter J23101(BBa_J23101), thus we can use red fluorescence as a detection.
Experimental approach
To wrap the E. coli into the alginate gel beads, we first weigh 4g of sodium alginate, add 100ml of hot water, mix well, put in an autoclave, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until the bubbles disappear. Then, Take 10 g of E. coli with tagRFP transformed wet cells, suspend them in 10 ml of sterile physiological saline, and prepare a bacterial suspension. After that, we slowly add the above sodium alginate solution and stir to make a suspension. The above suspension was gradually dropped into 1000ml of 0.2mol/LCaCL2 solution with a syringe with a 9-gauge needle and stirred with an electromagnetic stirrer while injecting to form bead-like particles (diameter 3ram). Finally, we soak in CaCL2 solution for 2h, then weigh 4g of sodium alginate and add 100ml of hot water, mix well, put in a high-pressure sterilizer, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until gas retention disappears.
Proof of express
After We transformed this plasmid into E. coli and encapsulated the E. coli into alginate gel beads, we then induced it to express tagRFP. We photograph the cross-section of the gel beads under a confocal microscope and get Figure 1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 985
Reference
[1] 赵景联.固定化大肠杆菌细胞生产γ-氨基丁酸的研究[J].生物工程学报,1989,5(02):124-128.DOI:10.13345/j.cjb.1989.02.010.
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