Part:BBa_K2797013

High copy BioBrick assembly plasmid pSB1C3 with RFP internal standard

pSB1C3 with a constituitively expressed RFP construct under the control of a medium strength Anderson promoter.

Usage and Biology

Newcastle iGEM 2018

In summer 2018, [http://2018.igem.org/Team:Newcastle/InterLab Newcastle iGEM GeneMachine] designed a constitutive, medium strength RFP expression construct and cloned said construct into the backbone of the iGEM InterLab test device pSB1C3 vectors - in the non-coding region between the chloramphenicol resistance gene and the Origin of Replication - for use as an Internal Standard within the iGEM 2018 InterLab study.

The addition of this RFP Internal Standard within the InterLab study devices revealed that, even under the same experimental conditions, the complex nature of biological systems still cause variation in protein expression, and highlights how important the use of an Internal Standard is in the identification of variation in part characterisation studies.

Major Findings

• Lower target gene expression - Fluorescein/OD regarding GFPmut3b was lower in pSB1C3 modified with the RFP Internal Standard construct with reference to those without.
• Transcription/translation machinery saturation - The stronger promoters (Test Devices 1 and 4) showed little to no fluorescence on the basis of RFP, despite sequencing revealing the presence of the gene. It is thought that these promoters are so strong that the addition of any other protein expression genes cause the saturation of transcription/translation mechanisms.
• Variation in protein expression - In devices were RFP fluorescence can be observed, there is significant variation in the expression of the gene despite the same experimental conditions, indicating that protein expression may be more complex than originally first thought.
• Target gene expression consistency - The presence of the Internal Standard in pSB1C3, while causes target gene (GFPmut3b) expression to be lower, allows the target gene expression to be more consistent with little to no fluctuations in expression over 24 hours in respect to pSB1C3 target gene expression without the Internal Standard construct.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 989
  Illegal suffix found in sequence at 1
  Illegal EcoRI site found at 3052
  Illegal XbaI site found at 3067
  Illegal SpeI site found at 1905
  Illegal PstI site found at 1919
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 989
  Illegal EcoRI site found at 3052
  Illegal NheI site found at 1017
  Illegal NheI site found at 1040
  Illegal SpeI site found at 2
  Illegal SpeI site found at 1905
  Illegal PstI site found at 16
  Illegal PstI site found at 1919
  Illegal NotI site found at 9
  Illegal NotI site found at 995
  Illegal NotI site found at 1912
  Illegal NotI site found at 3058
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 989
  Illegal EcoRI site found at 3052
  Illegal XhoI site found at 2036
  Illegal XhoI site found at 2928
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 989
  Illegal suffix found in sequence at 2
  Illegal EcoRI site found at 3052
  Illegal XbaI site found at 3067
  Illegal SpeI site found at 1905
  Illegal PstI site found at 1919
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 989
  Illegal EcoRI site found at 3052
  Illegal XbaI site found at 1004
  Illegal XbaI site found at 3067
  Illegal SpeI site found at 2
  Illegal SpeI site found at 1905
  Illegal PstI site found at 16
  Illegal PstI site found at 1919
  Illegal AgeI site found at 1627
  Illegal AgeI site found at 1739
  
• 1000
  COMPATIBLE WITH RFC[1000]