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 and P_FLD1 transcription profiles, measuring the performance of kinetic parameters and input compatibility using the standardization principles of synthetic biology [1]. We also tested some P. pastoris grow and cultivation variables to verify how it would behave on detection conditions and on the aforementioned lyiophilization (freeze-drying) process.

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

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.

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 <p>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 <a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">Assemblies page</a>.</p>
-<h4>Defining the best cryoprotectants </h4>
-		<p>As the baker’s dry yeast may confirm, protocols for yeast cells are abundant. Using as reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeast species[22], we tested combinations of two cryoprotectants for the lyophilization of <i>P. pastoris</i>: powdered milk and monosodic glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>). After realizing the lyophilization process with 1.5 mL tubes, the following results were obtained:
-			<p class="table">
-			<b>Table 2:</b> CFU for each test in 1.5 mL tubes. 24h lyophilization process with 1 mL of cell suspension.</p>
-			<table>
-				<tr class="tr_first"><td style="width:10%"></td><td style="width:20%">Glutamate</td><td style="width:20%">Milk + Glutamate</td><td style="width:20%">Milk</td><td style="width:20%">Control (only YPD) </td><td style="width:20%">CFUi</td></tr>
-				<tr><td>CFU/mL</td><td>8.8 x 10<sup>2</sup></td><td>7.76 x 10<sup>6</sup></td><td>4.4 x 10<sup>3</sup></td><td>0</td><td>3.667 x 10<sup>7</sup></td></tr>
-				<tr><td>%CFUi</td><td>2,4 x 10<sup>-5</sup></td><td>21</td><td>1,2 x 10<sup>-4</sup></td><td>0</td><td>100</td></tr>
-			</table>
-<p>Although threalose was not used&#8212;what is also a good protectant for yeast species [22]&#8212;, the result showed itself interesting enough for a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [23], this might not affect P<sub>AOX1</sub> 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 <i>Pichia</i> 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</a>.</p>