Team:Aachen/Interlab Study

From 2014.igem.org

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===Inoculation and Cultivation===
===Inoculation and Cultivation===
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The cultivation of our bacteria was performed in 500 ml shake flasks filled with 50 ml [http://2014.igem.org/Team:Aachen/Notebook/Protocols#LB_medium LB medium]. The cultures were kept at 37 °C and 300 rpm shaking frequency. Appropiate antibiotics were added to each media (kanamycin for I20260, chloramphenicol for B0015, J23101.E0240 and J23115.E0240). Both antibiotics were added from a 1000X stock stored at -20 °C for a final concentration of 35 µg/ml chloramphenicol and 50 µg/ml kanamycin, respectively.
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The cultivation of our bacteria was performed in 500 ml shake flasks filled with 50 ml [http://2014.igem.org/Team:Aachen/Notebook/Protocols#LB_medium LB medium]. The cultures were kept at 37°C and 300 rpm shaking frequency. Appropiate antibiotics were added to each media (kanamycin for I20260, chloramphenicol for B0015, J23101.E0240 and J23115.E0240). Both antibiotics were added from a 1000X stock stored at -20°C for a final concentration of 35 µg/ml chloramphenicol and 50 µg/ml kanamycin, respectively.
The precultures were inoculated from the same cryo stocks. They were cultivated for 16 hours and then sampled for OD measurement with a spectrophotometer. Then 2 ml of each preculture were centrifuged (5 minutes, 6000 g) and then washed twice with PBS buffer. Afterwards, all cultures were inoculated to have the same starting OD. Inoculations were carried out under sterile conditions at the clean bench.
The precultures were inoculated from the same cryo stocks. They were cultivated for 16 hours and then sampled for OD measurement with a spectrophotometer. Then 2 ml of each preculture were centrifuged (5 minutes, 6000 g) and then washed twice with PBS buffer. Afterwards, all cultures were inoculated to have the same starting OD. Inoculations were carried out under sterile conditions at the clean bench.
===Sampling===
===Sampling===
-
To draw samples, the shake flasks were taken out of the 37 °C room and brought onto a nearby bench. 3 ml of sample were taken out next to a Bunsen burner flame and pipetted into three 2 ml cuvettes. As soon as all samples were taken the flasks were taken back onto the shaker in the 37 °C room. The whole process of taking samples for all 12 flasks (3 biological replicates for each construct) took 5 minutes and samples were taken every 2 hours.
+
To draw samples, the shake flasks were taken out of the 37°C room and brought onto a nearby bench. 3 ml of sample were taken out next to a Bunsen burner flame and pipetted into three 2 ml cuvettes. As soon as all samples were taken the flasks were taken back onto the shaker in the 37°C room. The whole process of taking samples for all 12 flasks (3 biological replicates for each construct) took 5 minutes and samples were taken every 2 hours.
After 4 hours, we had to dilute the samples with LB medium in a ratio of 1:4, and from the 6th to 18th hour we had to dilute in a ratio of 1:9.
After 4 hours, we had to dilute the samples with LB medium in a ratio of 1:4, and from the 6th to 18th hour we had to dilute in a ratio of 1:9.

Revision as of 10:44, 17 October 2014

Interlab Study

As our team is competing in the Measurement track for this year's competition, we were also required to participate in the iGEM 2014 Measurement Interlab Study. This study aims to collect data from iGEM teams all over the world on the fluorescence of three genetic devices expressing GFP. The devices differ in their plasmid copy properties and the strength of the promotor.

We introduced the three constructs into E. coli cells and measured fluorescence as well as optical density of the liquid cultures over a period of 18 hours, using a spectrophotometer and a plate reader, respectively. The obtained results confirmed our hypothesis that the fluorescence of the BioBrick in the high copy plasmid pSB1C3, J23101.E0240, would exhibit a stronger signal than the constructs I20260, which is on the low to mid copy plasmid pSB3K3, and J23115.E0240, which has a weaker promotor than J23101.E0240. During the experiment, we could observe a typical growth curve for E.coli including lag, exponential, stationary and death phase. We could show that the fluorescence we measured is rather a function of each cell than the whole culture, since all cultures had comparable optical densities.


Aachen 14-10-13 Yellow Flask iNB.png

Experimental Design

For the Interlab Study, we tested GFP-containing BioBricks for fluorescence and optical density. Subject of the study were the BioBricks I20260, J23101.E0240 and J23115.E0240. The latter consists of a pSB3K3 backbone with an insert, a combination of the promoter J23101, the RBS B0032, the GFP coding sequence E0040 and the terminator B0015. J23101.E0240 has the same insert as I20260, but has pSB1C3 as a backbone. J23115.E0240 only differs from J23101.E0240 in the use of another promotor, namely J23115. As a negative control, we used just B0015 in pSB1C3.

Flasks.png

Aachen 14-10-11 BioBricks for Interlab iNB.png
Genetic devices tested
Composition of I20260, J23101.E0240 (left) and J23115.E040 (right)

Over a time span of 18 hours the optical density and fluorescence of cultures containing these BioBricks were measured every 2 hours using the spectrophotometer and plate reader, respectively.

Aachen 14-10-13 Green Flask iNB.png

Expected Results

Fluorescence was expected to develop in cultures containing I20260, J23101.E0240 and J23115.E0240, as all include the GFP coding sequence. However, the signal was expected to be stronger in J23101.E0240 than in I20260 since pS1C3 is a high copy plasmid while pSB3K3 is a low to mid copy plasmid. Because of this, a higher fluorescence was expected of J23101.E0240 compared to I20260 even though they share the same insert. J23115.E0240, too, was supposed to produce a fluorescent signal, but J23115 (the mutated version K823012 was used) is a lot weaker promotor than J23101. Therefore, a lot lower - if any - fluorescence is expected with this BioBrick.

Aachen 14-10-16 Plasmid Promotor Strength iNB.png
Diagram illustrating the different plasmid and promotor properties
Plasmid pSB1C3 has a higher copy number than pSB3K3, and J23101 is a stronger promotor than J23115.

B0015 was used as our negative control as the insert only contains a terminator and no expression cassette for GFPmut3b, and so no fluorescence was expected.

Aachen 14-10-13 Empty Falcon iNB.png

Materials and Methods

Constructs and strains

All constructs used were transformed into NEB 10β cells. The constructs I20260 as well as B0015 were taken directly from the iGEM 2014 distribution plates. The constructs J23101.E0240 as well as J23115.E0240 were made using the 3A Assembly. Therefore, the subparts J23101, J23115 as well as E0240 were transformed directly from the 2014 distribution plates into NEB 10β cells. Afterwards the plasmids were recovered using the illustra plasmidPrep Mini Spin Kit. The purified plasmids J23101 and J23115 were cut with the restriction enzymes EcoRI and SpeI, while E0240 was cut with XbaI and PstI. The restricted plasmids were then ligated together using the T4 DNA Ligase. Afterwards, the ligation product was introduced into the pSB1C3 linearized backbone provided by iGEM headquarters with the 2014 distribution which we had also cut with EcoRI and PstI. All restrictions and ligations were performed using enzymes and buffers of the NEB iGEM Kit. The final product was once again transformed into NEB 10β cells.

The correct identity of the resulting constructs were confirmed by sequencing. The sequencing data (consensus sequences) can be found here.

Note: We used the mutated version of J23115 as sent out by the iGEM headquarters. The mutation makes J23115 effectively the same promoter as K823012. We will still refer to the promoter as J23115 though, to keep it more easily recognizable with the other Interlab Study results.

Inoculation and Cultivation

The cultivation of our bacteria was performed in 500 ml shake flasks filled with 50 ml LB medium. The cultures were kept at 37°C and 300 rpm shaking frequency. Appropiate antibiotics were added to each media (kanamycin for I20260, chloramphenicol for B0015, J23101.E0240 and J23115.E0240). Both antibiotics were added from a 1000X stock stored at -20°C for a final concentration of 35 µg/ml chloramphenicol and 50 µg/ml kanamycin, respectively.

The precultures were inoculated from the same cryo stocks. They were cultivated for 16 hours and then sampled for OD measurement with a spectrophotometer. Then 2 ml of each preculture were centrifuged (5 minutes, 6000 g) and then washed twice with PBS buffer. Afterwards, all cultures were inoculated to have the same starting OD. Inoculations were carried out under sterile conditions at the clean bench.

Sampling

To draw samples, the shake flasks were taken out of the 37°C room and brought onto a nearby bench. 3 ml of sample were taken out next to a Bunsen burner flame and pipetted into three 2 ml cuvettes. As soon as all samples were taken the flasks were taken back onto the shaker in the 37°C room. The whole process of taking samples for all 12 flasks (3 biological replicates for each construct) took 5 minutes and samples were taken every 2 hours.

After 4 hours, we had to dilute the samples with LB medium in a ratio of 1:4, and from the 6th to 18th hour we had to dilute in a ratio of 1:9.

After the measurement of OD in the spectrometer, 100 µl of each sample were taken out and put on a 96-well plate (Thermo microfluor 1, flat-bottom, black) to measure fluorescence.

Each measurement occured in a technical triplicate, resulting in 36 different samples being processed in every sampling step.

Measurement of OD using a spectrophotometer

For OD measurement, the Unico Spectrophotometer 1201 of Fisher Bioblock Scientific was used. The measurement was taken at 600 nm and we used pure LB medium (from the same batch as the medium used for cultivation) as our blank. We only measured OD up to an absorbance of 0.8. At a higher OD, we diluted the sample with LB medium (again from the same batch as our cultivation medium). Dilution was done by first filling the cuvettes with the LB medium, and then adding our cultivation sample. Subsequently, each cuvette was vortexed thoroughly. The solution was allowed to settle before measurement.

Measurement of fluorescence using a microplate reader

Measurement of fluorescence was performed using the Synergy Mx from BioTek with the Gen5 software, using the following parameters:

Parameter value
Software version 2.1.2014
Reader Type Synergy Mx
Read GFP 100
Measurement fluorescence endpoint
Measurement range full plate
Filter filter set 1
Excitation 496 ± 9.0 nm
Emission 516 ± 9.0 nm
Gain 100
Read Speed normal
Delay 100 msec
Measurement s/data point 10
Read height 8 mm

As for the OD measurement, we used LB medium as our blank. Since samples for fluorescence measurement were acquired from the cuvettes for the OD measurement, sample processed in plate reader had the same dilutions.

Aachen 14-10-13 Graph Panel iNB.png

Results

After observing the optical density (OD) and fluorescence for 18 hours while taking samples every 2 hours, the following results were obtained:

Aachen 14-10-13 InterlabGraph1 iFG.PNG
Interlab Study Results
Our measurements of fluorescence and optical density of the three genetic devices and a negative control.

This shows that all cultures had ODs in the same range throughout the experiment. After the exponential growth phase the stationary phase started shortly after 4 hours of cultivation time. The OD did not change from thereon until a cultivation time of 16 hours after which it started to decline.

The development of fluorescence followed largely the pattern of the OD, but differed a lot in between the different cultures. J23101.E0240 exhibited fluorescence three times stronger than I20260, and about 10 times stronger than B0015 and J23115.E0240. The latter two did not differ in terms of fluorescent signal.

Aachen 14-10-13 Graph with Error Bars Panel iNB.png

Discussion

The OD is an indirect measurement of the biomass in the shake flask. Through the correlation of both measurements the results show that the difference in biomass of the cultures is not significantly enough to affect the fluorescence data. Therefore, the fluorescence data can be interpreted as a direct result of the fluorescence per cell instead of an overall fluorescence per culture.

The fluorescence data shows a strong difference between the I20260 and J23110.E240. Even though both inserts are the same, there is a difference in fluorescence, as expected, because of the different plasmid backbones. The high copy plasmid pSB1C3 shows a 3 times stronger fluorescence signal per cell than the low to mid copy plasmid pSB3K3. This can be directly related to the number of plasmids in the cells coding for GFP.

Both J23115.E0240 and B0015 show no significant fluorescence. The increase at 4 hours is explained by the increase of OD resulting in noise. B0015 behaves therefore as expected. J23115.E0240 in its original, non-mutated state was supposed to show a slight but weaker fluorescence than J23101.E0240. However, the mutations introduced made the promoter non-functional, which lead to no expression of GFP and therefore no observation of fluorescence.