Difference between revisions of "Part:BBa K3739050"

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===Biology===
 
===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.
 
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.
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===Usage===
 +
Here, we use BBa_3739020 to construct the expression system of LCIKR-2.
  
 
===Characterization===
 
===Characterization===
 
====1. Identification====
 
====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).
 
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).
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"https://static.igem.org/mediawiki/parts/2/2b/T--XMU-China--K3739050.png"
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https://static.igem.org/mediawiki/parts/2/2b/T--XMU-China--K3739050.png
 
::'''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).
 
::'''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).
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====2.Proof of the expression====
 
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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.
 
  
 +
====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.
  
 +
https://static.igem.org/mediawiki/parts/7/77/T--XMU-China--K3739050P.png
  
 
'''Fig. 2.''' SDS-PAGE analysis of LCIKR-2-GFP. Target bands of LCI KR-2-GFP (blank arrow, 33.2 kDa).
 
'''Fig. 2.''' SDS-PAGE analysis of LCIKR-2-GFP. Target bands of LCI KR-2-GFP (blank arrow, 33.2 kDa).

Latest revision as of 20:02, 21 October 2021


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). T--XMU-China--K3739050.png

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.

T--XMU-China--K3739050P.png

Fig. 2. SDS-PAGE analysis of LCIKR-2-GFP. Target bands of LCI KR-2-GFP (blank arrow, 33.2 kDa).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 263