Difference between revisions of "Part:BBa K4195152"
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===Biology=== | ===Biology=== | ||
| − | GFP | + | <b>GFP</b> |
The GFP derived from jellyfish ''Aequeora Victoria'' (<partinfo>BBa_E0040</partinfo>) is designed by team Antiquity in 2004. It’s a commonly used reporter. | The GFP derived from jellyfish ''Aequeora Victoria'' (<partinfo>BBa_E0040</partinfo>) is designed by team Antiquity in 2004. It’s a commonly used reporter. | ||
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====2. ''In Vitro'' Verification==== | ====2. ''In Vitro'' Verification==== | ||
Plasmid and linear DNA segments were put into the cell-free system for expression. The expression behavior of GFP is observed by measuring the fluorescence/OD<sub>600</sub> using microplate reader.<br/> | Plasmid and linear DNA segments were put into the cell-free system for expression. The expression behavior of GFP is observed by measuring the fluorescence/OD<sub>600</sub> using microplate reader.<br/> | ||
| − | [[File:T--XMU-China--T7-GFP-T7t.png|300px]] | + | [[File:T--XMU-China--T7-GFP-T7t.png|300px]]<br/> |
Fig. 2 The expression yield of linear DNA segments and circular DNA ''in vitro''<br/> | Fig. 2 The expression yield of linear DNA segments and circular DNA ''in vitro''<br/> | ||
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Revision as of 20:40, 10 October 2022
T7-GFP-T7t
Biology
GFP
The GFP derived from jellyfish Aequeora Victoria (BBa_E0040) is designed by team Antiquity in 2004. It’s a commonly used reporter.
Usage and Design
GFP was selected for its good performance in enhancing the report signal. We add BBa_K3222000, BBa_K4195068, BBa_B0034 and BBa_K731721 to construct the expression system and obtained the composite BBa_K4195152, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
Characterization
1. In Vivo Verification
1) Agarose Gel Electrophoresis
After transferring the plasmid into BL21(DE3), colony PCR was used to certify the plasmid was correct. The expected result was obtained (1182bp).
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Fig. 2 The result of colony PCR. Plasmid pSB1C3
2) Bioluminescence measurement Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of GFP is observed by measuring the fluorescence/OD600 using microplate reader. Results are documented in related pages: BBa_K4195168, BBa_K4195169, BBa_K4195170, BBa_K4195171, BBa_K4195172, BBa_K4195173, BBa_K4195174, BBa_K4195175, BBa_K4195183, BBa_K4195184, BBa_K4195185, BBa_K4195186, BBa_K4195187, BBa_K4195188, BBa_K4195189, BBa_K4195190.
2. In Vitro Verification
Plasmid and linear DNA segments were put into the cell-free system for expression. The expression behavior of GFP is observed by measuring the fluorescence/OD600 using microplate reader.
Fig. 2 The expression yield of linear DNA segments and circular DNA in vitro
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 823
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 38
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 739