Team:USP-Brazil/Results
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Results
Overview
Besides designing a bioengineered microorganism that could help solve the methanol poisoning
problem, the pragmatically goal on wet lab was to generate new data on P
The main questions regarding on our experimental approach are:
- BioBrick Characterization Questions
- How strong are the AOX1 and FLD1 promoters?
- How relatively strong are the PAOX1 variants?
- How different alcohol sources will behave on interaction with wild PAOX1 and variants? (Activation? Repression?)
- Will PFLD1 be activated or repressed by inputs other than mentioned on literature (methylamine and methanol)?
- How long is the time delay response of these promoters in a system with RFP?
- Have our Pichia codon-optimized RFP a better fluorescence than a non-optimized (BBa_E1010)?
- Microbiology Questions
-
Will P. pastoris survive on ethanol solutions?
Will P. pastoris survive lyophilization? If survives, will the survival population be large enough to deliver the output signal at naked eye!?
Our team managed to answer some of these questions and is still working to completely deliver all of them with good quality. You may start checking our results on our Assemblies page.
Defining the best cryoprotectants
As the baker’s dry yeast may confirm, protocols for yeast cells are abundant. Using as reference the known methodologies for Saccharomyces cerevisiae and other yeast species[22], we tested combinations of two cryoprotectants for the lyophilization of P. pastoris: powdered milk and monosodic glutamate (see our Notebook). After realizing the lyophilization process with 1.5 mL tubes, the following results were obtained:
Table 2: CFU for each test in 1.5 mL tubes. 24h lyophilization process with 1 mL of cell suspension.
Glutamate | Milk + Glutamate | Milk | Control (only YPD) | CFUi | |
CFU/mL | 8.8 x 102 | 7.76 x 106 | 4.4 x 103 | 0 | 3.667 x 107 |
%CFUi | 2,4 x 10-5 | 21 | 1,2 x 10-4 | 0 | 100 |
Although threalose was not used—what is also a good protectant for yeast species [22]—, the result showed itself interesting enough for a cheaper way to make lyophilized Pichia. Since lactose is not metabolized by this yeast [23], this might not affect PAOX1 activation if the powdered milk does not have residual glucose quantities. Those interesting findings support the next subject of our project: the idealized device who could compartmentalize the Pichia powder in a simple, secure and user-friendly way. Check the next section where we try to put all the pieces together to built the product.
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