Part:BBa_K4195117
I0500-B0034-his-linker-GFP-B0015
GFP is used to verify whether the outer-membrane vesicles can conduct horizontal gene transfer. 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.
Usage
The circuit is used to verify whether plasmids could be passed from outer-membrane vesicles (OMVs). If the transformation was done, the engineering bacteria can produce green fluorescence protein after being induced by L-arabinose. We used both BBa_I0500 and BBa_B0015 to construct the expression system and obtained this circuit, which are assembled on the expression vector pUC57-Simple 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 we need 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 beween 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. b </b>Solid medium plate of agar spot test containing BBa_K4195117.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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