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 Team Newcastle] 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. This is an improvement on the pSB1C3 plasmid which is usually used for these constructs.

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

Data from the RFP Internal Standard experiments is shown in figure 1, and from this data the following conclusions were gained.

• Lower target gene expression - Fluorescein/OD regarding GFPmut3b was lower in pSB1C3 modified with the RFP Internal Standard construct with reference to those without.
• 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.
• 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.

Figure 1. A) Scatter plots showing the relationship between Relative AFUs (GFPmut3b fluorescein/OD) against time (x-axis) for the original test device and the internal standard test devices. The legend for each device is shown on the left of each graph. Over 24 hours, the original test devices reach peak fluorescence at around the 6-7 hour mark, with devices 4 & 1 being the strongest and 3 & 6 being the weakest. After the 6 hour mark there is a decrease in fluorescence to the 22 hour mark where fluorescence beings to increase once again. The internal standard devices all showed a consistent increase with no decrease over 24 hours. Device 2 was the most fluorescent with devices 1 and 4 being significantly lower than the original group. Fluorescence was lower for each device initially than the original but eventually surpassed the original study as the fluorescence decreased over time. B) Scatter plots showing the relationship between Relative AFUs (RFP fluorescence/OD) (y-axis) and time (x-axis) for the RFP internal standard test devices. The legend for each device is shown on the left of the graph. Over the 24 period, the control groups and devices 1 & 4 exhibited a fluorescence/OD of 0. The remaining other devices - 2, 3 & 6 – all showed a consistent positive increase in fluorescence/OD. Device 2 however has the highest fluorescence/OD throughout the study, followed by devices 6, 3 and 2 with fluorescence/OD values of 0.064, 0.048 and 0.038 respectively. These groups began fluorescing between 200 and 440 min.

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]