Part:BBa_K808025:Experience

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Applications of BBa_K808025

This part was improved in 2013 by the iGEM team of the TU Darmstadt. Please take a look at BBa_K1055007 to see the improvement. Among others an online expression detection method was implemented to gain better control over the enzyme production.

User Reviews

UNIQfa516e240ce1cb57-partinfo-00000000-QINU UNIQfa516e240ce1cb57-partinfo-00000001-QINU

Applications of BBa_K808025

This part was used by Tianjin 2015 in its Stimulated Plastic Enzymolysis.We aim to enhance the effect of FsC by the addition of hydrophobin and make them into a fusion protein. We could provide abundant data about its expression in E.coli. You could also find its fusion protein in BBa_K1582020, BBa_K1582021,BBa_K1582022 and BBa_K1582023.

Protein Expression

Protein pre-expression FsCUse pET-28a as expression vector and pre-express FsC, FsC-sJanus, FsC- sJanus-m in BL21. Analyze the result of SDS-PAGE to infer whether protein FsC, FsC-sJanus, FsC-sJanus-m express after induced by IPTG.

Figure 2.The results of SDS-PAGE. M is Protein marker. a is sample of FsC which is non-induced. b is sample of FsC which is induced.

We can tell the difference (in red circle) between non-induced and induced. The amount of protein after induced is larger than non-induced and molecular weight is about 25kDa which is target protein’s molecular weight. So we infer that FsC can express in E.coli BL21.
·Inoculation of LB-media with BL21_ FsC _pET-28a; Overnight incubation at 37°C
·Dump cultivated bacteria into 1L liquid LB medium. Cultivate for 4h at 37°C. Add 1M IPTG 500μL and induce at 16°C for 15h.
·Centrifugation of 1L LB medium at 4000rpm for 20min and discard supernatant.
·Crush bacteria with High Pressure Homogenizer.
·Centrifugation at 18000rpm for 30min.
·Purify protein with nickel column.

Figure 2.The result of protein BL21_FsC expression. M is Protein marker. a is sample of FsC in BL21 which is non-induced. b is sample of FsC in BL21 which is induced. c is sample of supernatant after breaking bacteria and centrifugation d is sample of sediment after breaking bacteria and centrifugation. e if sample of liquid after filtration by Ni column. f is sample of media after filtration by Ni column. g is sample of liquid after removing impurity with 50mM MCAC. h is sample of media after removing impurity with 50mM MCAC. i is sample of target protein after washing with 200mM MCAC. j is sample of media after washing with 200mM MCAC.

There is still a little protein combine with media after washing with 200mM MCAC.
Nanodrop measurements of the purified protein FsC: 0.414mg/mL
·Concentrate FsC with concentration tube and change the buffer from 200mM MCAC to 1X PBS because 200mM MCAC is noxious for enzyme. Final concentration is 2.870mg/mL