Team:UGA-Georgia/RBS

Ribosome Library

The ribosome binding sites of archaea are not well characterized. Creating and characterizing a RBS library will be the first of its kind, allowing researchers the ability to express proteins of interest at variable levels of expression in Methanococcus.

Characterization of regulatory sequences – mCherry as a quantitative fluorescent reporter

To characterize the variability of expression per RBS, we must use some sort of quantitative approach. Use of the red fluorescent protein, mCherry, along with the quantitative measurements of a plate reader were our choices for characterizing our library of sequences. It’s important that we picked a red fluorescent protein because methanogens are naturally auto-fluorescent in the blue-green range, due to Coenzyme F420.

Last year, we created a first-draft vector (BBa_Kxxxxxxxx) that contained mCherry in the pAW50 vector. The largest issue with this construct was that the mCherry gene contained an internal Pst1 site, therefore making it BioBrick incompatible. Fluorescence readings from this construct were notably unreproducible, which will be elaborated on in the novel protocol for fluorescence reading section below. To improve upon this construct, we fixed the internal Pst1 site of mCherry to establish BioBrick compatibility and picked up a new constitutively expressing vector highly optimized for use in M. maripaludis, pMEV4.

The pMEV4-mCherry vector (fig. X) shown below is the construct we’ve designed to create and characterize our library of RBS’s. The primary differences between this vector and the pAW50-mCherry vector of 2013 is the internal restriction site has been removed from the mCherry gene, and the Puromycin resistance gene has an independent promoter. These fixes are particularly useful as this vector is now BioBrick compatible, and more reliably able to function under selective pressure. The region labeled ‘RBS 1-39’ is the 12 base-pair region including the first base of the start codon and the 11 bases immediately prior which will be subject to mutation. Primers were designed for a mutation on each base along a 12 base-pair mutation region (fig X). This region includes the RBS (1-18), spacer (19-33), and first base of the start codon (34-36). Additionally, two primers were designed (37 & 38) as theoretical ‘perfect’ and ‘negative’ RBS sequences. The theoretical ‘perfect’ and ‘negative’ RBS sequences (hereafter referred to as 37 & 38, BBa_Kxxxxxx & BBa_Kxxxxxx, respectively) were derived based off of 16S rRNA data found on “paper, author.” We chose to include these sequences in our library as effective positive and negative controls of RBS binding. The sequence in fig X labeled ‘Native RBS’ (hereafter referred to as Native, BBa_Kxxxxxxx) is a known functional RBS in methanogens that primarily, and usually solely, is used for creating synthetic parts. To characterize every sequence of the library, our approach is to first create three pools of libraries; 1) All RBS region mutation sequences, 2) All spacer region mutation sequences, and 3) All start codon mutation sequences. Anaerobic transformation in M. maripaludis is both time-consuming and meticulous, so by pooling libraries we reduce the number of transformations that need to be done. After picking a statistically appropriate number of colonies from the transformants to maximize likelihood of including every sequence, we will allow all of the cultures to grow under optimal growth conditions, then quantify fluorescence of mCherry using a plate reader. Then we will have all of the clones sequenced and correlate the sequence to the relative strength of fluorescence. However, we encountered some issues when it came to quantifying mCherry production from M. maripaludis that hasn’t been addressed in any previous literature which led to the development of the novel mCherry fluorescence quantification protocol for production in M. maripaludis, as described below.