Difference between revisions of "Part:BBa K3771079"

Revision as of 03:34, 19 October 2021

T7-JJU

Description

P_T7-jju is a composite part consisting of the T7 promoter and the cs sequences. This part was used in in vivo testing of taurine production. The sequence for jju and T7 promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

T7 promoter constitutively facilitates the expression of JJU enzyme.

Usage

JJU10 enzyme was used in vitro testing of taurine production. The sequence for JJU10 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC). A 6xHis-tag is added to the C-terminal of the JJU10 protein, allowing for confirmation of JJU10 expression by western blot using the anti-6xHis antibody.

Characterization

Fig 1. Confirmation of jju fragment by PCR. M: Marker; Lane 1: jju (1180bp)

Fig 2. Confirmation of pET28a fragment by digestion. M: Marker; Lane 1: pET28a (~5280 bp)

Fig. 3 Transformation/JJU in DH5α

Fig. 4 Confirmation of pET28a-T7-JJU by digestion. M: Marker; Lane 1: Colony of pET28a-T7-JJU (~6466 bp)

Fig. 5 Transform into BL21(DE3)

Fig. 6 Confirmation of protein expression of JJU. M: Marker; Lane1: whole cell of JJU in BL21(DE3); Lane2: soluble protein of JJUBL21(DE3) (~43 kDa)

Fig. 7 Transform into BD7G

Fig. 8 Confirmation of protein expression of JJU. M: Marker; Lane1: whole cell of JJU in BD7G; Lane2: soluble protein of JJU in BD7G(~43 kDa)

References
BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10
Sequence and Features BBa_K3771079 SequenceAndFeatures

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 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-<img src="https://2021.igem.org/wiki/images/e/e6/T--NCKU_Tainan--jju-plate%28BL21%28DE3%29%29.png" style="width:35%;">
+<img src="https://2021.igem.org/wiki/images/1/19/T--NCKU_Tainan--jju-PAGE%28BL2%28DE3%29.png" style="width:35%;">
 </div>
 <p align="center">Fig. 6 Confirmation of protein expression of JJU.
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 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--jju-plate%28BD7G%29.png" style="width:35%;">
+<img src="https://2021.igem.org/wiki/images/3/32/T--NCKU_Tainan--jju-PAGE%28BD7G%29.png" style="width:35%;">
 </div>
 <p align="center">Fig. 8 Confirmation of protein expression of JJU.