Part:BBa_K4195152

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).

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 823
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 38
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 739