Difference between revisions of "Part:BBa K4195116"
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===Characterization=== | ===Characterization=== | ||
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Agarose Gel Electrophoresis When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target bands(2256 bp) (Fig. 1)at the position between 2000bp and 3000bp. | Agarose Gel Electrophoresis When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target bands(2256 bp) (Fig. 1)at the position between 2000bp and 3000bp. | ||
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| + | [[File:T--XMU-China--LMT sp-his-GFP colony PCR band.png|400px]] | ||
<b>Fig 1. DNA gel electrophoresis of the colony PCR products of <partinfo>BBa_K4195116</partinfo>_pUC57-Simple.</b> | <b>Fig 1. DNA gel electrophoresis of the colony PCR products of <partinfo>BBa_K4195116</partinfo>_pUC57-Simple.</b> | ||
Revision as of 09:43, 11 October 2022
I0500-B0034-LMT sp-his-linker-GFP-B0015
LMT is a signal peptide from the lytic murein transglycosylase of V. natriegens, his-tag enable us to purify the linking protein.
LMT here represents a signal peptide used to secrete the protein to the periplasm. 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
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.
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.
LMT sp
Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kinds of LMTs existing in E. coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of Vibrio natriegens and E. coli.
Characterization
identification
Agarose Gel Electrophoresis When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target bands(2256 bp) (Fig. 1)at the position between 2000bp and 3000bp.
Fig 1. DNA gel electrophoresis of the colony PCR products of BBa_K4195116_pUC57-Simple.
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