Characterizing BBa_K2117000

Literature-reviewed characterization

Molecular elements for Y. lipolytica such as strong constitutive promoters have not been well established in the iGEM registry. In an effort to add to the limited library of Y. lipolytica parts in the iGEM registry, we characterized the important TEF1 promoterBBa_K2117000 using the information available in the literature. TEF1 is a native promoter of the Translation Elongation Factor 1 (TEF1) gene in Y. lipolytica . TEF1 is considered to be one of the strongest native promoters found inY. lipolytica . This was confirmed when expression of the reporter gene, hrGFP, under TEF1 promoter and several other strong endogenous Y. lipolytica promoters were tested and TEF1 was shown to have one of the highest expression levels among the native promoters.

Despite TEF1 showing high expression levels, a stronger promoter might be required for some engineering purposes such as metabolic engineering. Therefore, we identified methods of improving TEF1’s expression levels using tandem copies of upstream activation sequences 1B (UAS1B). Fusion of 16 copies of UAS1B to the 5’ promoter core element of the TEF1 promoter significantly enhanced the strength of the promoter. The strength of the promoter could also be further increased by fusing a truncated form of TEF1 promoter with the UAS1B copies. This is due to the fact that closer proximity of the enhancer element to the core promoter element could help improve its function.

Future Characterization

We used the TEF1 promoter in a few of our genetic constructs (BBa_K3629015 and BBa_K3629018) as we needed a strong constitutive promoter to express our cellulase genes. However, we are unsure if the activity of the TEF1 promoter varies depending on the growth stage of the yeast, which would affect all recombinant protein production. Therefore, we decided to characterize the growth-phase dependent activity of the TEF1 promoter BBa_K2117000.

PART DESIGN

This part was based on the design of BBa_2117005 with the hrGFP, and was ordered from IDT with an XRP2 terminator added at the end.

EXPERIMENTAL DESIGN

Figure 2 shows the experimental workflow that will be followed to characterize this part in the future upon greater lab access

Figure 2. Experimental workflow of how BBa_K2117000 will be experimentally characterized in the future. The activity of the TEF1 promoter in Yarrowia lipolytica will be characterized based on growth state to determine how growth phase may impact promoter activity.

1. Make a 4mL overnight culture with YPD media and a wild-type Y. lipolytica colony and grow overnight at 30ºC
2. Make a 3 replicate 1:50 dilutions with YPD media with 1mL each of the overnight culture. Grow shaking at 28ºC
3. Sample under fluorescence plate reader with 488 nm laser excitation and 510 nm emission, as well as OD 650, after 6, 12, 18, 24, and 30 hours
• Samples at 6, 12, and 18 hours are expected to correspond with log phase. Samples at 24 and 30 hours are expected to correspond with stationary phase.

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REFERENCES

Next Steps

In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.

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