Difference between revisions of "Part:BBa K2117005"

Revision as of 23:57, 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 (1).

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).

Figure 1: Analytical digestion of the pSB1A8YL containing the this TEF1-hrGFP device. The fragment lengths can be seen on the ladder, and the restriction enzyme and predicted fragment lengths is stated above the fragments.

Afterwards the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy. Figure 2 shows the Y. lipolytica PO1f cells under a confocal laser microscope with 100x magnification. The high fluorescent output from this construct, proves that the cells are producing hrGFP, and ultimately that our expression system consisting of pSB1A8YL (BBa_K2117009) and the TEF1 promoter (BBa_K2117000) can be used for heterologous protein expression in Y. lipolytica.

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 this GFP expressing device shuttled by our plasmid pSB1A8YL, (BBa_K2117009). 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.

References: 1) Blazeck, J., Liu, L., Redden, H., Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. APPLIED AND ENVIRONMENTAL MICROBIOLOGY.