Difference between revisions of "Part:BBa K4115044"

Latest revision as of 02:47, 5 October 2022

J23101-B0034-tagRFP-B0015

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.

We first transformed this composite part into the plasmid backbone PUC57 to verify that it could express tagRFP protein. Later, we will transform it into the plasmid backbone pBBR1 and transfer it into A.caulinodans ORS571 as a shuttle plasmid to mark A.caulinodans ORS571.

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 and Figure 2.

Figure 1: 10X Confocal image of cross-section of gel beads with red fluorescent protein E. coli.

Figure 2: 63X Confocal image of cross-section of gel beads with red fluorescent protein E. coli.

Sequence and Features

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.