Coding

Part:BBa_K2656020

Designed by: Adrián Requena Gutiérrez, Carolina Ropero   Group: iGEM18_Valencia_UPV   (2018-09-23)


YFP_LVA Coding Sequence

domestication.
Figure 1. DNA basic parts domestication. Third construction corresponds with CDS Basic Part adaptation into the GoldenBraid grammar.

Part BBa_K2656020 is the Yellow fluorescent protein with LVA degradation tag sequence compatible with both Biobrick and GoldenBraid 3.0 assembly methods. It can be combined with other compatible parts from our Valencia UPV IGEM 2018 Printeria Collection to assemble transcriptional units with the Golden Gate assembly protocol . We designed this part to perform our improvement project:

First, we adapted the CDS BBa_K592101 to be used to assemble composite parts using the Golden Gate method, creating BBa_K2656021 and we added the LVA degradation tag, creating BBa_K2656020, our improved part. Next, we performed an experiment to obtain the excitation and emission spectra. To do this, we created the transcriptional unit BBa_K2656112 and we used the parameters of the Table 1:

Table 1. Parameters used to obtain the spectra

Parameter Value
Number of samples 6
Excitation Wavelength measurement range (nm) [450-550]
Emission wavelenght (nm) 580
Emission Wavelength measurement range (nm) [500-580]
Excitation wavelenght (nm) 470
Gain (G) 50


YFP spectra.
Figure 2. YFP emission and excitation spectra

To test the effect of the degradation tag, we designed an experiment with which we measured the increase in protein degradation due to this tag. To perform this experiment, we assembled two composite parts with the same promoter, RBS and terminator:

Once the experiment was carried out, the results were plotted and Figure 3 was obtained, in which we can observe that the growth of the bacteria with both constructions was very similar, while the fluorescence had a clear variation.


sfGFP spectra.
Figure 3. Experimental results of the fluorescence comparison experiment between the transcriptional unit with BBa_K2656021 and the one with BBa_K2656020.

These data were optimized with our model and the parameters from Table 2 were obtained. With these parameters it is possible to obtain that the degradation of the protein with the tag is around twice as much as the degradation of the protein without the tag.

Table 2. Optimized values of translation rate, degradation rate and dilution rate from experimental data

Optimized parameters

Values

Translation rate p

PoI degradation rate dp

Dilution rate μ


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter/yfp
//function/reporter/fluorescence
Parameters
colorYellow
directionForward
emissionYFP
emit529
excite516
proteinYFP
tagLVA