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.

To further characterize this commonly used Y. lipolytica promoter, we gathered data on the performance of individual components of the TEF1 promoter such as the proximal promoter element (PPE), the TATA box, and the upstream activation sequence (UAS). Upon the removal of PEE, TEF1 was shown to remain functional but its strength was significantly reduced. Removal of the TATA box, however, completely diminished promoter function. Both the TEF1 TATA box and the PPE were shown to have one of the highest affinities for transcription factors in Y. lipolytica . Thus their inclusion in building hybrid promoter significantly increased promoter strength. We also found that the addition of TEF1 UAS upstream of expression construct was shown to significantly boost expression levels.

We hope that our in-depth characterization of the Y. lipolytica TEF1 promoter can make it easier for future iGEM teams to work this new exciting chassis. Using the information we provided, teams can make a more informed decision on their choice of promoter. Moreover, based on the characterization information provided on individual promoter components, teams can decide on how to play around with promoter composition to obtain their desired expression levels.

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 XPR2 BBa_K3629004" terminator added at the end.

EXPERIMENTAL DESIGN

Figure 3 shows the experimental workflow that will be followed to characterize this part in the future upon further lab access.

Figure 3. 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.

Protocol is as follows:

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.