Part:BBa_K2239010
CBD-GFP
CBD-GFP (T7 promoter--lac operator--RBS--His-tag--GFP--CBD--T7 terminator)
This device codes for the GFP-CBD fusion protein.
Construct
The vector of GFP-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 GFP sequence is retrieved from the GenBank. It is artificially synthesized and inserted into plasmid pUC57. The GFP 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 GFP 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--GFP--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-GFP]
Usage and Biology
This part is used to test the function of CBD (cellulose binding domain), which is able to bind to cellulose.[1]
Expression
The constructed pET28x-GFP-CBD 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.
Proof of the CBD function
The resulted solution after expression is mixed with a gauze piece, and the green color on gauze is recorded. Then the gauze is washed three times using ddH2O, but the green fluorescent on it is not significantly reduced, 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]
Reference
[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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 990
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 751
Illegal SapI site found at 846
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