Part:BBa_K2943902

Promoter, RBS, mRFP (Improved)

This part is an improvement of an existing composite part: https://parts.igem.org/Part:BBa_K608014.
It has a native constitutive promoter, and contains improvement of Shine-Dalgarno(SD) sequence(including inside an improved RBS) and improvement of the monomer RFP (mRFP). The goal of the improvement was to enable more efficient and stronger expression of mRFP through improving both the mRNA folding energy and through improvement of the SD that has direct impact on translation initiation and speed.

The Experiment:
In order to check if our improvement worked we have designed an fluorescence experiment to compare between the original composite part and our version. Using 96 plate reader, we measured each part fluorescence intensity. In addition we also added wells for negative control (Bacteria with non fluorescence plasmid) and LB for blank. We have repeated the the measuring a few times through the day to make sure we do not get any false results and to make sure the bacteria had enough time to grow and express the protein. The bacteria concentration was measured in OD700 to avoid any false measuring due to the absorption of light by the mRFP.

Fluorescence was measured using the data below:
OD: 700nm
Excitation- Monochromator
Excitation wavelength: 540nm
Excitation bandwidth: 50nm
Emission- Monochromator
Emission wavelength: 650nm
Emission bandwidth: 20nm
Gain: 103 Optimal

Results(Fig.1):
Negative Control Mean- 26,766.72; STDEV- 3423.982
Original Part Mean- 57,853.17; STDEV- 2624.755
Our Part Improvement Mean- 154,336.4; STDEV- 8994.965

The results above were received by the next 2 step calculation:
1. Calculating for each well the intensity:
Individual Intensity= (Florescence of plasmid measure by the machine - florescence of LB) / # Colonies in OD700
2. Calculating the average of individual intensity for each plasmid.

This part have shown stronger intensity by almost 3 time that the original part its based off!

Reference:
[1]. Gross, Larry A., et al. "The structure of the chromophore within DsRed, a red fluorescent protein from coral." Proceedings of the National Academy of Sciences 97.22 (2000): 11990-11995.
[2]. Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms for Molecular Biology 6, 26 (2011)
[3]. Hecht, Ariel, et al. "When wavelengths collide: bias in cell abundance measurements due to expressed fluorescent proteins." ACS synthetic biology 5.9 (2016): 1024-1027.
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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 617
  Illegal AgeI site found at 729
  
• 1000
  COMPATIBLE WITH RFC[1000]