Team:USP-Brazil/Results:Assemblies

Results

Assemblies

To explore the questions on BioBricks characterization, we built the construction of the following strains:

Figure 1: Map of planned test strains. DNA construction images hiding RBS and transcription stop sequences.

The P_AOX1 strains are basically different combinations of variants of only three DNA elements: the P_AOX1 promoter, the RFP reporter protein and the modified Mxr1 transcription factor (aforementioned on Detecthol). Thanks to Life Technologies, the strain B1 and the Mxr1 modified and minimum were synthesized and this enabled us to construct the others strains.

Comparing the fluorescence time delay and intensity between strains Control 1, A1, and B1, we will be able to check the strength and the response velocity of the device with modified P_AOX1 and the efficiency of codon-optimization on RFP for P. pastoris. The comparison between control strains 2 with A2 and B2 will draw the picture of P_AOX1 promoter behavior with the modified Mxr1 transcription factor with the same variables from the last comparison, but also testing if the shorter version of Mxr1—consisting in the sequence for the N-terminal 400 amino-acids from Mxr1—cited on literature [2] will work properly.

To assembly those parts we: used the strain B1 as template to built the strain A1 using PCR (polymerase chain reaction) and restriction enzymes; the control strain 1 was assembly in the pPIC9K plasmid, which was used to transform P. pastoris, allowing to integrate the construction by homologous recombination in the genome; the strains A2, B2 and Control strain 2 are made by transforming P. pastoris with the corresponding P_AOX1-RFP construction and the Mxr1 plasmid. … gel… o que a gente conseguiu construir, o que está na linha de produção... e etc.

Transformation

Characterization

Pichia growth on ethanol solutions

To test the promoters response to the inputs, we did quick preliminary tests with P. pastoris to evaluate it survival ability to ethanol concentrations to define a specific ethanol range for input testing.

Figure 3: Growth curve of Pichia pastoris. Gray x mark the actual data; colored circles represents the mean and the line is the fitted logistic curve. Both curves represent the same conditions, but starting the measuring with two different initial ODs.

After making a growth curve of Pichia pastoris on simple YPD media (graph above), we defined the OD of the stationary phase. With this information we tested the yeast growth repression in presence of ethanol, measuring samples in different ethanol concentration solutions when the control sample (without ethanol) achieved the stationary phase. The results are following:

Figure 4

Some were also grow on YDP plates to qualitatively evaluate their viability:

4.5% Ethanol	6% Ethanol
7.5% Ethanol	9% Ethanol

Figure 5

This result shows us that, besides stagnation on growth, the cells remain viable. Further tests relating to this subject were done and could be found hereafter on lyophilization results. For now, this information is enough to proceed with the characterization of our planned strains, knowing that the cells resist ethanol percentages normally found in alcoholic beverages.

Measuring Input Intensity Response from Strain Control 1

With the first successful transformation on Pichia pastoris, the Control 1 Strain (see transformation results), we ready measured the cells response on a range of concentration of methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters for mRFP1 fluorescence assay present on BBa_E1010 information. We obtained the graph below for samples from the same cultivation, starting with DO 0.1:

Figure 6

This preliminary result indicates that exist an inferior limit of P_AOX1 expression induction (equal or lower than 50 mM). This is still not conclusive, because represents only the first hours of yeast induction. Repetitions of this test will be very clarifying about the behavior of this promoter on methanol induction. This kind of analysis will enable to characterize the promoter with great precision. These data will be very important to the comprehension of promoter’s function and validation of our mathematical model.

{Aqui entra depois uma parte bem crucial para dizermos que as coisas estão rolando e que quase estamos com mais resultados legais que nem esse. Infelizmente já estou cansado e sem inspiração pra coisas importantes como essa, vou deixar em aberto também, desculpa, pessoal!}