Difference between revisions of "Part:BBa K1618032"

Latest revision as of 16:16, 21 September 2015

Acyltransferase Candidate 4 -Fused to GFP -Expression Construct

K1618030 (P-35s(K1467101)_TP(K1618037):RV3037c(K1618004):GFP(K1467204)_T-35s(BBa_K1618037)) expresses a putative acyltransferase fused to a C-terminal fluorescent reporter in plant cells and targets the gene product to the chloroplast.

The parts were assembled using Golden Gate Assembly and the assembly was tested in a binary plasmid vector backbone prior that can replicated in the shuttle chassis Agrobacterium tumefaciens that transfers the part to plant cells on a T-DNA. For submission to the registry the part was converted to a standard BioBrick by using the Mo-Flipper BBa_K1467400 (complete transcriptional unit flipper) made by the NRP-UEA 2014 team.

Functional characterisation in the model plant Nicotiana benthamiana (Figure 1) indicates that the chloroplast transit peptide is functional and is able to direct the enzyme to the chloroplast where starch is produced.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 392
Illegal BglII site found at 1138
Illegal BamHI site found at 2341
Illegal XhoI site found at 1342
Illegal XhoI site found at 3476
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2151
Illegal NgoMIV site found at 2324
Illegal NgoMIV site found at 2648
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K1618032 short</partinfo>
-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):RV3037c(K1618004):GFP(K1467204)_T-35s(BBa_K1618037). The construct should be able to acylate starch in plants.
+K1618030 (P-35s(K1467101)_TP(K1618037):RV3037c(K1618004):GFP(K1467204)_T-35s(BBa_K1618037)) expresses a putative acyltransferase fused to a C-terminal fluorescent reporter in plant cells and targets the gene product to the chloroplast.
-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 [https://parts.igem.org/Part:BBa_K1467400 BBa_K1467400 ] (complete transcriptional unit flipper) made by the NRP-UEA 2014 team.
+The parts were assembled using Golden Gate Assembly and the assembly was tested in a binary plasmid vector backbone prior that can replicated in the shuttle chassis <i>Agrobacterium tumefaciens</i> that transfers the part to plant cells on a T-DNA. For submission to the registry the part was converted to a standard BioBrick by using the Mo-Flipper [https://parts.igem.org/Part:BBa_K1467400 BBa_K1467400 ] (complete transcriptional unit flipper) made by the NRP-UEA 2014 team.
+Functional characterisation in the model plant <i>Nicotiana benthamiana</i> (Figure 1) indicates that the [https://parts.igem.org/Part:BBa_K1618028 chloroplast transit peptide] is functional and is able to direct the enzyme to the chloroplast where starch is produced.
-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.