Part:BBa_K4195014
his-linker-GFP
GFP is used to verify whether the outer-membrane vesicles can conduct horizontal gene transfer.
Usage
In order to make bond with secondary antibody to detect the produce of GFP as a signal showing successful transformation, we added a his-tag (6*His) at the N-terminal of GFP. And to reduce the possibility that the his-tag may affect the function of GFP, we added a linker between his-tag and GFP. Then we assemble the inducible promoter (BBa_I0500), the part (his-linker-GFP) and the double terminator (BBa_B0015) on the expression vector pUC57-Simple to get the composite part BBa_K4195117 by standard assembly. The constructed plasmids were transformed into E. coli DH5α and BL21(DE3), then the positive transformants were selected by ampicillin and confirmed by colony PCR and sequencing. After a successful construction was confirmed, we carried out outer membrane (OMVs) extraction to obtain OMVs to incubate with Vibrio alginolyticus.
Biology
GFP
A gene codes for a green fluorescence protein. Whether the fluorescence of GFP presents or not after inducing is our standard to confirm the success of incubation caused transformation from OMVs to Vibrio alginolyticus.
Characterization
Identification
Agarose Gel Electrophoresis
When we were constructing this circuit, regular PCR was used to certify the plasmid was correct (Fig. 1). We got the target bands(2199 bp) at the position between 2000 bp and 3000 bp.
Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195117_pUC57-Simple.
OMVs extraction and plasmids packaging verification
The colony with the correct sequence was cultivated to extract OMVs by hypervesiculation. After extraction, some OMVs were used to PCR to verify if there were target plasmids packaging in. The target bands (Fig. 2)(2199 bp) were observed at position between 2000 bp and 3000 bp.
Fig. 2 Verification of OMV’s ability to load plasmid. a Solid medium plate of agar spot test. b The result of OMV PCR. Contained plasmid is BBa_I0500_pSB1C3.
Fig. 3 Agar spot test of OMVs. a Solid medium plate of agar spot test containing b Solid medium plate of agar spot test containing BBa_K4195117.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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