Difference between revisions of "Part:BBa K4195015"
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<partinfo>BBa_K4195015 short</partinfo> | <partinfo>BBa_K4195015 short</partinfo> | ||
| − | + | LMT is a signal peptide from the lytic murein transglycosylase of <i>Vibrio natriegens</i>, 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. | ||
| + | |||
| + | ===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 (<partinfo>BBa_I0500</partinfo>), the part (LMT sp-his-linker-GFP) and the double terminator (<partinfo>BBa_B0015</partinfo>) on the expression vector pUC57-Simple to get the composite part <partinfo>BBa_K4195116</partinfo> by standard assembly. The constructed plasmids were transformed into <i>E. coli </i>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 <i>Vibrio alginolyticus</i>. | ||
| + | |||
| + | 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 <i>E. coli</i>: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of <i>Vibrio natriegens</i>. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of <i>Vibrio natriegens</i> and <i>E. coli</i>. | ||
| + | |||
| + | |||
| + | ===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 2000 bp and 3000 bp. | ||
| + | |||
| + | [[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>. Target bands can be observed at the position between 2000 bp and 3000 bp. | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 10:20, 13 October 2022
LMT sp-his-linker-GFP
LMT is a signal peptide from the lytic murein transglycosylase of Vibrio 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.
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 (LMT sp-his-linker-GFP) and the double terminator (BBa_B0015) on the expression vector pUC57-Simple to get the composite part BBa_K4195116 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 2000 bp and 3000 bp.
Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195116_pUC57-Simple. Target bands can be observed at the position between 2000 bp and 3000 bp.
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]