Team:UNIPV-Pavia/Methods Materials/Absorbance

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Absorbance and water dispensation

We find no relevant differences between the measures before and after the dispensation of different volumes of water in the well. As regards liquid growth medium these differences are practically null, in the case of bacterial coltures, instead, there is a positive trend, but the values vary into a negligible interval.

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Test%2017_10_09/ Download Protocol] - Test 17/10/09.

Absorbance and water dispensation, LB

Absorbance and water dispensation, bacterial colture

Absorbance and volume

The proportionality between absorbance (OD600) measurement and volume of colture in the well was verified. By fitting a linear regression model on the experimental data we estimate a coefficient of proportionality of 8.27e-4 for bacterial cultures and of 1.38e-4 for LB+Amp medium.

You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/10_10_09test9bis/ Download Protocol] - Test n.9bis, 10/10/09 (bacterial colture), and You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Test%2017_10_09/ Download Protocol] - Test 17/10/09 (LB medium).

Absorbance and volume (0-300ul), bacterial colture

Absorbance and volume (0-300ul), LB medium

Absorbance and dilutions in liquid growth medium

The proportionality between absorbance measurement (OD600) and culture's dilutions was verified, confirming the possibility of using OD600 to measure the bacterial quantity in the well, regardless of the total volume present in the well.

This experiment was done in two different growth media, LB and M9 supplemented + glycerol, and in three different total volumes in the well, 100μl, 200μl and 300μl.

By fitting a linear regression model on the experimental data shown in the two figures below, we estimate a coefficient of proportionality of 0.0018 for LB medium and 0.0035 of for M9.

You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.20, 13/08/09 and Test n.20 "M9", 28/08/09.

Absorbance and culture's dilutions in LB

Absorbance and culture's dilutions in M9

Different protocols for different growth curves

As our first step of standard measurements in Synthetic Biology, we wanted to individuate a proper protocol to have reproducible growth curves for the cultures incubated inside the microplate reader.

We compared three different solutions, as you can see in the figures below.

Protocol 1:

• overnight culture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
• dilution 1:1000;
• incubation 37°C 220 rpm for about 2/3 hours;
• use this colture to fill the plate.

Protocol 2:

• pick a colony from a streaked plate with single colonies;
• infect the microplate well, previously filled with 200 μl of growth medium.

Protocol 3:

• overnight culture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
• dilution 1:100 or 1:1000;
• incubation 37°C 220 rpm for about 2/3 hours;
• infect the microplate well, previously filled with 200 μl of growth medium, with a sterile pipette tip poured in the grown culture.

Growth Protocols n.1 and n.2

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.5, 18/06/09.

As you can see in the figure above, the protocol n.2 does not give any reproducibility of growth curves, and sometimes doesn't guarantee the observation of all growth phases because of the noise in the first part of the curve, probably due to high density matter or solid medium taken from the plate that interfere with absorbance measurements. Moreover, this procedure allows to use the same clone taken from a colony just once, without the risk of loosing bacteria during the infection operation.

On the other hand, protocol n.1 ensures a perfect reproducibility of the curves coming from the same falcon tube. Clearly cultures from different falcon tubes evolve in different way.

For all these reasons we decided to consider this protocol for our experiments.

Growth Protocol n.3

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.6, 27/06/09.

The protocol n.3, as you can see in the figure above, allows the complete observation of the curves, but not their reproducibility. Moreover, it takes a very long time to reach O.D. values significantly different fom 0. For these reasons, it was discarded.

Growth ambients in comparison

A 10-hour experiment was made to compare the growth in the microplate (200 ul of culture) with those in typical growth ambients like 50 ml falcon tube (30 ml of culture) and 500 ml flask (200 ml of culture), especially in the exponential phase. The goal was the estimation of important parameters that characterize growth curves, as duration and evolution of all the phases, and doubling time in exponential phase.

As you can see in the figures below, the growth inside the microplate is well comparable to the other two till the end of its exponential phase (4 hour, estimation done on the logarithm of the blanked data), then slowes down, becoming almost linear, to reach O.D. values lower than the others (0.4). At the end of the exponential phase the O.D. measurement is 0.14 and the doubling time estimate is 40 minutes (referring to the logistic growth model): these parameters are consistent with those present in literature.

As regards the other ambients, we observed a longer exponential phase (about 6 hours), with a final O.D. value of 0.46 for the flask and 0.51 for the falcon tube, and a doubling time of 55 minutes for both ambients.

This analysis allows us to individuate another important protocol to observe the exponential phase, which is the optimal growth phase and is very important for gene expression of sigma70 promoters. The starting point is protocol n.1, described in Different protocols for different growth curves, the new step is performing a dilution after the 2/3-hours incubation till reaching an O.D. value of about 0.02: wells filled with this diluted culture will be in the exponential phase for about two hours after the incubation and all the cultures will start to grow in the microplate at the same O.D.

Growth ambients in comparison

Growth ambients in comparison, exponential phase