From 2012.igem.org

Revision as of 15:47, 26 October 2012

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• Results and Devices
  • Strains characterization
  • Strains description
  • Fluorescence screening
  • Screening of strain proportion
  • Auxotrophy confirmation
  • Coculture in liquid medium
  • Revertant control
  • Trp basal production
  • Growth dependence

  • BBs design
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  Data Page

After the Jamboree!

When we returned from the LatinAmerican Jamboree we focused all our effords 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

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.

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

Figure X. check

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

After a few calculations..

Trp Secretion at different His concentrations in medium and His secretion at different Trp 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 K death

In order to determinate the K death used in the modeling section of our wiki, we set cultures of the two auxotrophic strains without being transformed (TCY3081 and TCY3128) 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.

TABLA CON EL NUMERO DE CELULAS POR DIA

Growth rate vs [TRP] and [HIS]

We made dilutions in series for increasing concentrations of Trp or His(1/32; 1/16; 1/8 ; 1/4; 1/2; 1X) in a -T or -H medium respectively. We made 2 replica of each concentration.

We set the cultures of the different strains at a low initial OD:0.001 approximately.

i. strain TCY3081 without device in each Trp concentration

ii. strain TCY3128 without device in each His concentration

iii. strain TCY3081 without device in Synthetic Complete medium

iv. strain TCY3128 without device in Synthetic Complete medium

v. Control (Synthetic Complete medium)

We measured OD reached by cultures 12 hs after started.

CONVERTIR ESTO EN UNA TABLA CON LOS RESULTADOS

Co-Culture of strains

We 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.