Difference between revisions of "Part:BBa K1618032"

Revision as of 20:09, 17 September 2015

Acyltransferase Candidate 4 -Fused to GFP -Expression Construct

This is an acyltransferase candidate with the aim to acylate starch. This version is fused to GFP, making it an expression construct. The construct is made up of P-35s(K1467101)_TP(K1618037):RV3034c(K1618003):GFP(K1467204)_T-35s(BBa_K1618037). The construct should be able to acylate starch in plants.

The construct was developed using GoldenGate Cloning. These parts were tested by cloning into a binary plasmid vector backbone prior to delivery to plants leaves using Agrobacterium tumefaciens mediated transfection. This was then converted to a standard BioBrick for shipping to the registry by cloning into the Mo-Flipper Plasmid BBa_K1467400 (complete transcriptional unit flipper) made by the NRP-UEA 2014 team.

NRP-UEA 2015 used this construct with a fluorescent tag to check the localization of plastid to chloroplast, as this is where starch is produced. We transformed our constructs into Agrobacterium tumefaciens and then infiltrated it into our plants. We used the YFP tagged constructs to confirm the localisation of parts to the chloroplast (Figure 1) and the untagged constructs to analyse the starch content of our leaves.

UEA_K1618032_confocal.jpg‎

Figure 1: Constructs BBa_K1618032 contains a fluorescent protein, as well as a chloroplast transit peptide. This is a confocal microscopy image of the construct infiltrated into Nicotiana benthamiana, in which the red structures are the chlorophyll within the chloroplast, and the yellow is the fluorescent fusion protein expressed from the construct.

The results above indicate that our expression construct is functional in confirming the localisation of our part into the chloroplast.

Sequence and Features