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Team:Buenos Aires/Results/BBsTesting
From 2012.igem.org
Contents |
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
Plasmids
| YFP_TRP+++_TRPZipper | details |
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| YFP_TRP+_TRPZipper | details |
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| YFP_TRP+++_PoliWb | details |
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| YFP_TRP+_PoliWb | details |
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| CFP_HIS+_PoliHa | details |
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| CFP_HIS+_PoliHb | details |
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Week 3: synthetic Ecology Characterization
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BB characterization
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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_TRP+++_TRPZipper
- YFP_TRP+++_PolyWb
- YFP_TRP+++
- YFP_TRP+_TRPZipper
- YFP_TRP+_PolyWb
- YFP_TRP+
- 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
Trp Secretion at different His concentrations in medium
We started cultures in -T of the following strains:
i. strain TCY3081+pCM185 containing device2 ii. strain TCY3081+pCM185 containing device4 iii. strain TCY3081+pCM185 (empty plasmid)
We measured the OD of the cultures after 12 hs. We prepared cultures with increasing concentrations of His in medium (1X, 1/4; 1/8; 0X)and set cultures at initial OD: 0.1. After 5 hours we measured the final OD and Trp in medium.
Strain characterization
Experimental determination of strains death rate
We set out to determine how long can auxotrophic 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, we'll 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 auxotrophic strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01.
- Taking into account that an OD:1 is approximately 3.10^7 cells/ml, we plated 133 µl in order to have around 200 colonies in the first day. Each following 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.
Table: Number of colonies counted per plate.
FOTOS DROPBOX Growth rate in function of the concentration of Trp and HisWe aimed to test the growth of the strains in different concentrations of external Trp and His in order to determine the amount of aminoacid at which the culture reaches half of the maximum growth (parameter K in the modeling of our system). We therefore made a series of cultures with increasing concentrations of Trp (Series 1)and His (Series 2). We set the cultures of the different strains at a low initial OD:0.001 with the following scheme: Series 1: strain YFP in each Trp concentration (dilutions 1/32; 1/16; 1/8; 1/4; 1/2; 1x) Series 2: strain CFP in each His concentration (dilutions 1/32; 1/16; 1/8; 1/4; 1/2; 1x) Controls: As control we started cultures of each strain in complete medium. Cultures were left overnight (12 hs) and we measured OD reached with the use of spectrophotometer.
ANALISIS DE VERO Co-Culture of strainsWe proceeded then to test the coculture growth of transformed strains with devices vs transformed strains with empty devices and non transformed strains.
Table: Coculture planification. Number indicates level of priority of the experiment for characterization of devices. Due to a time constrain, we ordered the experiments by level of priority and proceeded to test our devices by cocultures 1 and 2. Starters of each strain were done in medium complementary to the auxotrophy of the strain so that they would maintain their plamids (medium –H for pEG202; medium –T for pCM182/5 and medium SC for cells without plasmid), then sonicated briefly in low power and then washed with medium –H-T . When they are in exponential phase, we set the coculture of strains was at OD 600: 0.02 (Total, 1:1), in 5 ml of medium –H-T. We used epifluorescence microscope in order to determinate the strain proportion of each coculture. We used a 384 wells plate , with 20 µl of cyclohexamide 2x (CHX 2x) in each of the wells where we placed a sample. The density of the culture was calculated based on the cell density at in each wells.
Graph: 384 wells plate to be used for epifluorescence microscope.
Table: Coculture planification for number 1. |
