Team:UAlberta/Improve
VALIDATED CONTRIBUTION
amajLime is a yellow-green fluorescent protein that is used in various parts in the BioBrick Registry and has been successfully expressed and characterized by iGEM teams, including our own.
However, the amajLime-based parts submitted by previous teams do not include any tags for affinity-based purification which is a relatively accessible and routinely used for marker based studies. Team UAlberta's application of amajLime in the Beetector system is incompatible with amajLime obtained from cell lysates, unless purified. This is because our system relies on SortaseA (SrtA) to conjugate amajLime and M13 coat proteins together. Having an impure source of amajLime will decrease the efficiency of SrtA enzyme to modify the sortase tagged proteins and make the characterization of our reporter phage concentration inaccurate.
Inaccurate fluorescence and absorbance characterization within more than 5% margin of error will offset our Beetector reporter phage measurements in the modelling and application. Thus, Team UAlberta presents composite part BBa_K3072000 which is to be our validated part/contribution to the iGEM BioBrick Registry. This construct aims to introduce a polyhistidine tag (BBa_K1223006) on the C-terminus of the amajLime peptide (BBa_K1033916). We chose a polyhistidine as its function is very well characterized and our team had access to Ni-NTA purification columns and as polyhistidine has been used with fluorescent proteins similar to amajLime [1].
Currently, our team has ordered and designed the gBlocks and primers needed to construct the part. Our progress towards validating the part will be presented at the Jamboree and will include: cloning workflow and Gibson assembly using designed primers to incorporate the part into the pBAD vector [2]. All of our reporter proteins that are tagged with the C-terminal SrtA tagged (BBa_K3072510) are also designed with a C-terminal polyhistidine tag. Part BBa_K3072000 lacks a SrtA-tag and will be used as a positive control to compare amajLime characterization between His-tagged and SrtA His-tagged amajLime constructs.
References
- On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption
- Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Ai HW, Hazelwood KL, Davidson MW, Campbell RE. Nat Methods. 2008 May . 5(5):401-3. 10.1038/nmeth.1207 PubMed 18425137