Difference between revisions of "Part:BBa K3598000"

Revision as of 15:02, 24 October 2020

AOX1 promoter

AOX1 is a very common inducible promoter in yeast, and its ON/OFF condition is controlled by methanol.

Usage and Biology

We added AOX1 promoter and terminator into a plasmid in Pichia pastoris containing the sequence of sfGFP and recorded the intensity of the expression of fluorescence proteins in our experiment. We added 5% methanol into the culture for 5 days, and extracted the supernatant and ran 4%-20% SDS-PAGE gel for every 24 hours.

Performance of AOX1 in in Pichia pastoris

Characterized by Beijing_4ELEVEN 2020

In our project, we selected AOX1, a common inducible promoter used in Pichia pastoris GS115, as our promoter (BBa_I764001) to control the expression. To make sure the AOX1 promoter works properly, we tested its performance with sfGFP.

We bonded sfGFP sequences to the AOX1 promoter, inserted the whole sequence into pPIC9K backbone and transformed the plasmid into P. pastoris GS115. The recombinant strain was then verified of its fermentation in BMMY Medium. We measured the OD600 and fluorescence of the fermentation broth every 24 hours, and verified the supernatant samples during fermentation through SDS-PAGE gel electrophoresis.

Growth curve and fluorescence test

The OD600 of the recombinant strain containing sfGFP is a little lower than the control strain P. pastoris GS115, which might be the result of additional expression of sfGFP. The results show that expression of heterologous genes repress cell growth unintensively.

Figure 1. OD600 absorbance of P. pastoris GS115 and recombinant strain that contains sfGFP every 24 hours.

The fluorescence of recombinant strain that contains sfGFP becomes higher for every 24 hours, while that of P. pastoris GS115 remains mostly the same. This shows that AOX1 promoter under the control of methanol has the capability of promoting fluorescent protein expression.

Figure 2. Fluorescence of P. pastoris GS115 and recombinant strain that contains sfGFP every 24 hours.

SDS-PAGE gel analysis From the results (Figure 3), correct bands of sfGFP were shown as expected. The protein band at 24h can be barely seen; that at 48h is greater; and at 72, 96, 120h are nearly the same, all greater than at 48 h. So the clarity of the bands increases over time, which means the protein expression is getting more intensive every 24 hours. All the results prove that AOX1 promoter works properly.

Figure 3. SDS-PAGE gel analysis of supernatant samples during the fermentation

Source

Mycobacterium Phage Bxb1

References

Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S. & Lu, T.K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).

Sequence and Features