Team:NTNU Trondheim/Improve

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The RRvT’s strong brightness and it’s low sensitivity of acidic environment is supposed new biobrick a clear improvemt of the existing red fluorescent protein.

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Gold medal criterion #2, and best composite part award:

To improve a previous part, we decided to change the sequence of the red fluorescent protein coding device BBa_J04450. This was done by replacing the RFP sequence with the sequence for the RRvT. RRvT is a red fluorescent protein with a much higher brightness, and it can be used when the concentration of the inducer is to low to activate the BBa_J04450 biobrick. The RRvT has a very low acid sensitivity which can be useful in many situations. The advantages of RRvT’s strong brightness and it’s low sensitivity of acidic environment makes this protein better than the existing red fluorescent protein ....

The improved biobrick was synthesized by IDT as a linear sequence containing prefix and suffix. The prefix and suffix sequences can be found at parts.igem.org, and contain restriction enzyme cut sites that can be used to transfer and assemble parts. We then ligated it with the pSB1C3 (sjekk) backbone to make it RFC10 compatible. The improved biobrick contains a lactose induced promoter (Plac BBa_R0010), a ribosome binding site (BBa_B0034), the RRvT sequence and a terminator (BBa_B0015). The new biobrick is therefore a composite part. We have not sent the basic part with the RRvT sequence alone into the registry. Only the composite part was tested and sent in to the iGEM registry.

The testing of the new improved biobrick was done in parallel to the existing BBa_J4450 biobrick. The parts were measured ca every five minutes for 30 hrs by a Tecan plate reader. When the absorbance reached 0.8 Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to see if this gave any effect on the fluorescence. A growth curve for absorbance and fluorescence were then made. These curves were used to determine how the fluorescence changed with time.

The following figure shows growth curves for RRvT and mRFP as absorbance against time.

Fluorescence plotted against time for the two biobricks are shown in the table below. Citation: Wiens, M. D., Shen, Y., Li, X., Salem, M. A., Smisdom, N., Zhang, W., … Campbell, R. E. (2016). A Tandem Green-Red Heterodimeric Fluorescent Protein with High FRET Efficiency. ChemBioChem, 17(24), 2361–2367. doi:10.1002/cbic.201600492

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