Part:BBa_K3304100

Improved control vector with EYFP/ECFP for the expression strength measurement

Vector used for measurement of expression strength.

Designing and experimentally validating a foundational advance project that aims at a better understanding of the transcription factors’ binding to absolute DNA sequences, we accept the significance of deeply embedding the regulation of gene expression. Regarding the construction of regulatory devices, identification of the appropriate Promoter - RBS combination is of pivotal importance. Inspired by iGEM Bielefeld_CeBiTec 2018 team, who through testing the strength of a single promoter, a single RBS and their combination, designed a promoter-RBS library and a suitable measurement system to analyze the expression strength of the chosen promoter-RBS combination, we concluded in improving their part Part:BBa_K2638560 by choosing an advanced fluorescent protein match for the measurement.

It has already been stated that the measurement of a vector carrying two reporter genes is being conducted via normalization of the measured promoter - RBS’ strength expression to a constant expression level of the reference reporter gene. The fluorescent proteins that are going to be encoded by the aforementioned reporter genes ought to fulfill four criteria. Those criteria regard its bright fluorescence signal, its spectrally distinguishable excitation and emission, its similar maturation rates with the fluorescent protein tested and its similar DNA sequence close to the upstream promoter. Evaluating the alternative fluorescent proteins by these criteria, we concluded inserting in our construct the EYFP fluorescent protein whose emission and absorption spectra do not interact with their ECFP’s equivalents. Additionally, the EYFP and ECFP fluorescent proteins have very similar sequences as well as maturation half-lives.

Having mentioned the above, mRFP Part:BBa_E1010 used in the part introduced by Bielefeld_CeBiTec 2018 team appears to have apparent differences concerning its Fluorescence intensity through time when compared to ECFP, as stated by Rudge et al. 2016. Furthermore, considering the similarities that appear between ECFP and EYFP in Fluorescence intensity through time and cell growth, while also considering that EYFP's maturation time is lower than the mRFP equal, it is suggested that EYFP to be a more appropriate part to enter the construct sequence. However, due to time constraints, our team wasn't able to introduce experimental data on this hypothesis.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 936
  Illegal NheI site found at 959
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]