Difference between revisions of "Part:BBa K4907100"
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<partinfo>BBa_K4907100 short</partinfo> | <partinfo>BBa_K4907100 short</partinfo> | ||
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===Biology=== | ===Biology=== | ||
====GFP==== | ====GFP==== | ||
GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 533 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength (1). | GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 533 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength (1). | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | To test whether the two signal peptides LMT and KpSP whether can secret target proteins or not, this composite part was constructed to compare the fluorescence intensity. ===Characterization=== | + | To test whether the two signal peptides LMT and KpSP whether can secret target proteins or not, this composite part was constructed to compare the fluorescence intensity. |
| + | ===Characterization=== | ||
====Agarose gel electrophoresis (AGE)==== | ====Agarose gel electrophoresis (AGE)==== | ||
<partinfo>BBa_I0500</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_K4907036</partinfo> and <partinfo>BBa_B0015</partinfo> were assembled to obtain the composite part <partinfo>BBa_K4907100</partinfo>, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into <i>E. coli</i> DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. | <partinfo>BBa_I0500</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_K4907036</partinfo> and <partinfo>BBa_B0015</partinfo> were assembled to obtain the composite part <partinfo>BBa_K4907100</partinfo>, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into <i>E. coli</i> DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. | ||
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yyn/k4907100-fig1.png" width="400px"></html></center> | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yyn/k4907100-fig1.png" width="400px"></html></center> | ||
| − | <center>Fig. 1 Colony PCR of BBa_K4907100_pSB1C3 in <i>E. coli</i> DH10β | + | <center>Fig. 1 Colony PCR of BBa_K4907100_pSB1C3 in <i>E. coli</i> DH10β</center> |
====Fluorescence Intensity Ratio==== | ====Fluorescence Intensity Ratio==== | ||
The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> DH10βThis engineered bacteria with iE.coli</i> DH10β harboring <partinfo>BBa_K4907101</partinfo>_pSB1C3 and iE.coli</i> DH10β were cultivated and induced by 0.2% <i>L</i>-arabinose at 37 ℃ 12 hours. Then 1 mL bacterial liquid of each group was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As Fig. 2 shows, the fluorescence intensity of supernatant versus bacterial liquid of <i>E. coli</i> DH10βharboring <partinfo>BBa_K4907101</partinfo>_pSB1C3 was lower than the bacteria which express GFP extracellularly. | The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> DH10βThis engineered bacteria with iE.coli</i> DH10β harboring <partinfo>BBa_K4907101</partinfo>_pSB1C3 and iE.coli</i> DH10β were cultivated and induced by 0.2% <i>L</i>-arabinose at 37 ℃ 12 hours. Then 1 mL bacterial liquid of each group was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As Fig. 2 shows, the fluorescence intensity of supernatant versus bacterial liquid of <i>E. coli</i> DH10βharboring <partinfo>BBa_K4907101</partinfo>_pSB1C3 was lower than the bacteria which express GFP extracellularly. | ||
Latest revision as of 12:41, 12 October 2023
I0500-B0034-gfp-B0015
Biology
GFP
GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 533 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength (1).
Usage and Biology
To test whether the two signal peptides LMT and KpSP whether can secret target proteins or not, this composite part was constructed to compare the fluorescence intensity.
Characterization
Agarose gel electrophoresis (AGE)
BBa_I0500, BBa_B0034, BBa_K4907036 and BBa_B0015 were assembled to obtain the composite part BBa_K4907100, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
Fluorescence Intensity Ratio
The plasmid verified by sequencing was successfully transformed into E. coli DH10βThis engineered bacteria with iE.coli</i> DH10β harboring BBa_K4907101_pSB1C3 and iE.coli</i> DH10β were cultivated and induced by 0.2% L-arabinose at 37 ℃ 12 hours. Then 1 mL bacterial liquid of each group was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As Fig. 2 shows, the fluorescence intensity of supernatant versus bacterial liquid of E. coli DH10βharboring BBa_K4907101_pSB1C3 was lower than the bacteria which express GFP extracellularly.
Reference
- T. D. Craggs, Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 38, 2865-2875 (2009).
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