Difference between revisions of "Part:BBa K2239008"

Revision as of 00:06, 1 November 2017

CBD-LDH

CBD-LDH (T7 promoter--lac operator--RBS--His-tag--LDH--CBD--T7 terminator)

This device codes for the LDH-CBD fusion protein.

Construct

The vector of LDH-CBD for its expression is pET-28x. It is formed by modifying the restriction enzyme sites EcoR I and Xba I of vector pET-28a.

The LDH sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The LDH gene is then cloned from the plasmid by PCR amplification. The restriction site BamH I is added to the upstream primer, and Hind III is added to the downstream primer.

The CBD sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The CBD gene is then cloned from the plasmid by PCR amplification, with the restriction site Hind III added to the upstream primer, and Xhol I added to the downstream primer.

Firstly, the LDH gene is inserted into the modified pET-28x at BamH I and Hind III, and CBD at Hind III and Xhol I, after proliferation in T3 vector. Then the whole gene fragment, T7 promoter--lac operator--RBS--His-tag--LDH--CBD--T7 terminator, is retrieved from this plasmid by PCR amplification, with prefix containing EcoR I, Not I and Xba I added on its upstream primer, and suffix containing Pst I, Not I and Spe I added on its downstream primer. The PCR product is then connected to pSB1C3 at EcoR I and Pst I.

[Fig. 1. pSB1C3-CBD-LDH]

Usage and Biology

LDH(lactate dehydrogenase) catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back. It is used to catalyze the reduction of pyruvate into lactate, with the presence of NADH, which is oxidized into NAD+ at the same time.[1]

CBD (cellulose binding domain) is able to bind to cellulose. When connected to LDH, CBD is able to immobilize the enzyme LDH after expression, by binding to the gauze inside the solution on its cellulose.[2]

The function of cellulose binding domain

The function of CBD is tested by connecting CBD gene with GFP gene in pET28x. The GFP-CBD fusion protein is expressed and mixed with a gauze piece. The green fluorescent on the gauze is not significantly reduced after washing, proving that the CDB is well functioned. In comparison, no green fluorescent is left after washing the gauze mixed with GFP-ChBD (Chintin binding domain).

[Fig. 2. GFP-CBD on gauze before washing]

[Fig. 3. GFP-CBD on gauze after washing]

[Fig. 4. GFP-ChBD on gauze before washing]

[Fig. 5. GFP-ChBD on gauze after washing]

Expression and Immobilization[2]

The constructed pET28x-LDH plasmid is transformed into BL21(DE3) E.coli for expression. After that, when the OD 600 reached 0.6-0.8, 0.2mM IPTG is added in the liquid culture. The mixture is shaken at 20 ℃ overnight. The bacteria is collected by centrifugation at low temperature, 8000 rpm for 10 minutes, and the supernatant is discarded. The bacteria is then resuspended using 0.15M pH8.8 Tris-HCL, and is broken by ultrasonication.

The resulted bacteria solution is diluted to a certain concentration and mixed with gauze piece and the gauze piece is washed three times by ddH2O afterwards. As a result, the CBD protein binds to the cellulose on gauze, and the enzyme is successfully immobilized.

Reference

[1] Ming-Min Zheng, Ru-Feng Wang, Chun-Xiu Li, Jian-He Xu: Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochemistry, Elsevier, 2015.

[2] Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov: Immobilization of Recombinant Heparinase I Fused to Cellulose-Binding Domain, 1999.

Sequence and Features