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Revision as of 00:06, 27 October 2012

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After the Jamboree!

When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole.

We devided this task in three sections

Week 1&2 : Yeast expression vectors & Transformations

In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:

1. pCM182/5, which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996].
2. pEG202, with a 2 ori, HIS3 marker and a constitutive promoter (PADH1).

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The cloning we did was:

Protocol

1. Digestion of plasmids and TRP/HIS export device:
   1. pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.
   2. pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.
2. Purification of digested vectors (pCM185; pCM182; pEG202)
3. Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)
4. Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.
5. Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.
6. Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).

Once obtained the desired constructions, we transformed yeast strains:

1. TCY3081: W303, bar1-, ura3::PAct1-YFP
2. TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405

We got the following transformed strains:

TCY3081 (YFP) TCY3128 (CFP) Strain Name pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2 pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2 pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb

Week 3: Synthetic Ecology Characterization

Our task is to characterize our system including the devices functioning. We want specifically to:

1. Quantify the export of Trp as proof that the devices work
2. Determine experimentally some of the parameters that we used in the model
3. Characterize the growth of the transformed strains in coculture

In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.

Devices characterization

Secretion Rate of Trp as a function of culture growth

The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?

To test this we used the following strains:

• YFP_TRPb_TRPZipper2
• YFP_TRPb_PolyWb
• YFP_TRPb
• YFP_TRPa_TRPZipper2
• YFP_TRPa_PolyWb
• YFP_TRPa
• YFP

Protocol

• We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.
• Starting OD for the assay 0.1 (exponential phase).
• We measured OD every hour until they reached an OD: 0.8 (5 hs approximately).
• We measured the Trp signal for each culture medium using the spectrofluorometer.

We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take

After a few calculations, we find that

Next we show the average temporal evolution for each strains used,

Figure X. check

Tryptophan secretion at increasing histidine concentrations

We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system.

In this experiment we used strains:

- YFP_TRPb_TRPZipper2

- YFP_TRPb_PolyWb

- YFP_TRPb (control)

Protocol

1. Starters of each strain used were grown over night in -T media, at 30 °C in shaker.
2. After 12 hs, cells were pelleted and washed with -HT media.
3. Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.
4. We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.

Results

Strain characterization

Experimental determination of strains death rate

We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the death parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.

Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our system's feasability.

Protocol

• We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01.
• Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.

Strain Replica Monday Tuesday Wednesday CFP 1 260 320 285 CFP 2 267 314 76 CFP 3 413 362 278 YFP 1 230 316 688 YFP 2 291 194 524 YFP 3 449 344 725 Table: Number of colonies counted per plate. We expect to see a decrease in the number of colonies - because of cell death. We found that this was not the case, in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures. We can infer from this data that though they have not died, they may have enter into a ...Alan state. 100px 100px FigureX. Growth dependence on the Trp and His concentration An important thing characterize the system is the dependence of the growth rate of the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His). This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth). Protocol 1. We would use strains YFP and CFP (non-transformed, without devices). We started cultures of 5 ml of initial OD: 0.025 for: - Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x - Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x - Strain YFP at Synthetic Complete media - Strain CFP at Synthetic Complete media 2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer. Results To be done! Coculture of transformed strains We did the first experiment to test whether our system works and how two transformed strains grow together. We designed an assay to do so; following the growth of these strains: Transformed cells coculture and controls. Treatment Strain A Strain B 1 YFP_TRPb_TRPZipper2 CFP_HIS_PolyHa 2 YFP_TRPb_TRPZipper2 CFP_HIS 3 YFP_TRPb_TRPZipper2 4 YFP_TRPb CFP_HIS_PolyHa 5 CFP_HIS_PolyHa 6 YFP_TRPb CFP_HIS 7 CFP_HIS 8 YFP_TRPb Protocol Starters of strains were grown over night at 30°C, according the scheme showed in the table The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase. Cells were centrifugated and then washed with medium –HT. We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl. Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells. We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells. Strain Media YFP_TRPb_TRPZipper2 -T YFP_TRPb -T CFP_HIS_PolyHa -H CFP_HIS -H 384 wells plate to be used for epifluorescence microscope. Results