Part:BBa_K5401000

pUC_T7Promoter_eCFP

High-copy number plasmid encoding for enhanced cyan fluorescent protein (eCFP) under T7 Promoter.

The plasmid was utilised as part of a reporter system to evaluate the efficiency of T7 RNA polymerase and its variants.

Usage and Biology

T7 RNA Polymerase is a RNA polymerase derived from the T7 bacteriophage. It shows very high transcriptional activity, and is very specific to its cognate promoter: 5'-TAATACGACTCACTATAGG-3'. T7 RNA Polymerase is widely utilised in the field of biotechnology, such as in areas of in vivo protein expression and in vitro transcription.

The main purpose of this plasmid serves as a reporter, to evaluate the efficiency of the T7 RNA polymerase and other generated variants.

Characterization

The plasmid is first transformed into BL21 (DE3) E. coli cells, followed by colony PCR to confirm the successful transformation. A single colony is then inoculated for further culturing for IPTG induction. While initial results indicate that fluorescence was observed, further sub-culturing has resulted in the loss of fluorescence.

Fig 1: Quantification of relative fluorescence intensity for all three reporter systems. From left to right, C1(-IPTG), C1(+IPTG), C2(-IPTG), C2(+IPTG), C3(-IPTG) and C3(+IPTG) [LEFT] Replicate 1; n=8 technical replicates. [CENTER] Replicate 2; n=6 technical replicates. [RIGHT] Replicate 3; n=6 technical replicates.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2492
  Illegal suffix found in sequence at 17
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2492
  Illegal XbaI site found at 2507
  Illegal SpeI site found at 17
  Illegal PstI site found at 31
  Illegal NgoMIV site found at 1390
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2188