Difference between revisions of "Part:BBa K2117005"

Revision as of 16:32, 19 October 2016

Device encoding TEF1 and hrGFP for expression in Yarrowia lipolytica

This is a composite part consisting of the TEF1 promoter (BBa_K2117000) and hrGFP (BBa_K2117003).

This part encodes the humanized Renilla reniformis green fluorescent protein (hrGFP) codon-optimized for Yarrowia lipolytica and the native Y. lipolytica constitutive promoter TEF1.

Studies have shown the hrGFP to be functional in Y. lipolytica under the expression of the TEF1 promoter measured by flow cytometry. *insert reference*

Usage and Biology

The green fluorescent protein originates from the sea pansy, Renilla reniformis. This part can be used as a reporter protein for protein expression in the yeast chassis, Y. lipolytica. It is compatible with all the iGEM Biobrick standards.

Emission: 500-600 nm

Excitation: 488 nm

Sequence and Features

Functional Parameters

Emission: 500-600 nm

Excitation: 488 nm

Experimental data

The two parts (TEF1 and hrGFP) was assembled with the pSB1A8YL backbone (BBa_K2117009) using standard 3A assembly and transformed into E. coli. The construct was confirmed using restriction analysis (See Figure 1), PCR and sequencing (data not shown). The construct was then transformed into Y. lipolytica PO1f along with the pSB1A8YL plasmid without inserts as negative control.

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           <img id="img4" class="enlarge img-responsive figure-img" src="" alt="DESCRIPTION" width="150">
           <figcaption class="figure-caption">Figure 1: Analytical digestion of the pSB1A8YL containing the <a href="https://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a> device. The fragment lengths can be seen on the ladder, and the restriction enzyme and predicted fragment lengths is stated above the fragments.</figcaption>
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The transformation cultures were grown in 30 C in 48 hours and analysed by Confocal Laser Fluorescent microscopy. The control showed some autofluorescence, but the transformant with the construct gave a clear GFP signal (See Figure 2).

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           <img id="img3" class="enlarge img-responsive figure-img" src="" alt="DESCRIPTION" width="478">
           <figcaption class="figure-caption">Figure 2: Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using standard brightfield, B and E are taken using the GFP filter and with the excitation laser on and C and F are overlays of the two photos where the black bagground has been removed (C is an overlay of A and B, and F is an overlay of D and E). A, B and C are Y. lipolytica PO1f cells with our GFP expressing device (<a href="https://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>) shuttled by our plasmid pSB1A8YL. D, E and F are Y. lipolytica PO1f cells with the empty pSB1A8YL plasmid, which serves as a control for the GFP signal. Notice that even though the empty vector control shows trace amounts of auto-fluoresence the strain with the GFP expressing device clearly exhibits higher levels of fluorescence, which proves that our expression system works as intended.</figcaption>
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