Team:UAlberta/Contribution

VALIDATED CONTRIBUTION

amajLime Expression Construct (BBa_K3072999)

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_K3072999 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].

We have successfully constructed an amajLime expression construct that is arabinose-inducible via the araBAD promoter and the produced protein can be purified using affinity chromatography as the protein is tagged with a polyhistidine tag. We were also able to successfully express, extract, and purify the amajLime protein that resulted in greater yield and purity than the non-His-tagged version we were originally working with. Please see below for purification validation.

Figure 1:Extracted amajLime that is being purified using affinity based column chromatography (left) and the fluorescence measurements of the different fractions from the purification (right)

Overall, this part serves as both a validated contribution to the registry and improvement of previous amajLime parts as the protein can now be purified and is under the control of a very effective promoter system for protein expression.

References

1. 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
2. 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