Part:BBa_K3739050
J23100-B0030-LC1KR-2-GFP-B0010
LCIKR-2 is a mutant of a peptide called LCI from Bacillus subtilis, can sticks to polypropylene strongly. We use BBa_K3739050 to verify its capability by purification the protein.
Biology
LCIKR-2 is a mutant of a peptide called LCI from Bacillus subtilis, can sticks to polypropylene strongly.. GFP is green fluorescent protein from jellyfish Aequorea Victoria, which has been widely used as reporter for decades. GFP is fused at C terminal with LCIKR-2 so that the expression of LCIKR-2 will be reported.
Usage
Here, we use BBa_3739020 to construct the expression system of LCIKR-2.
Characterization
1. Identification
In order to verify whether the LCIKR-2-GFP was expressed accurately, LCIKR-2-GFP (BBa_K3739020) was assembled into the plasmid backbone (Fig. 1A). After being verified by agarose gel electrophoresis, the plasmids have been transformed into Vibrio natriegens successfully (Fig. 1B).
- Fig. 1. Gene circuit and agarose gel electrophoresis. (A) Gene circuit of J23100-B0030-LC1KR-2-GFP-B0010 (BBa_K3739050). (B) Target bands of LCIKR-2-GFP (black arrow, 1200 bp).
2.Proof of the expression
After successful construction, the plasmid was transformed into Vibrio natriegens through electroporation. The single positive colony was cultivated in 10 mL LB medium with chloramphenicol (working concentration was 12.5 μg/mL). After ultrasonication broke and centrifugation, the crude protein was released to the supernatant. After being verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel (Fig. 2), the protein of LCIKR-2-GFP was successfully expressed.
Fig. 2. SDS-PAGE analysis of LCIKR-2-GFP. Target bands of LCI KR-2-GFP (blank arrow, 33.2 kDa).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 263
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