iGem_BIT

Interlab Study

Summary

The Interlab study aims at collecting the data of three specific GFP (BBa_K823005+BBa_I13504, BBa_K823008+BBa_I13504 and BBa_K823013+ BBa_I13504)expression devices measured by iGEM teams all over the world, thus to promote the business of standardized measurement.
Our Interlab work was carried out with the DH5-alpha as an expression host, and the Thermo Scientific Varioskan Flash spectral scanning as the equipment. We transfer the three devices into E.coli cells separately. And we measure the fluorescence data and optical density data with spectral scanning machine every 1 hour during a 16 hours cultivation. And we also apply the dilution operation to the sample considering the OD interference.

Methods

We use endonuclease (EcoRI and PstI, XbaI and PstI) and T4 DNA ligase (NEB) to construct them. Finally, the ligation product was introduced into the pSB1C3. All constructs used were transformed into DH5α cells.
The cultivation of our bacteria was performed in tubes filled with 7 mL LB liquid medium. The cultures were kept at 37 °C and 110 rpm in shaker.
Antibiotics were added to each media (chloramphenicol 102μg/ml for K823005. I13504, K823008. I13504 and K823013. I13504).
We used100mL Luria Broth Liquid media(102μg/mL chloramphenicol) in 250 mL conical flask to grow our cells(37℃，110rmp). Every 1h, we took 200μL in 96-well plate (round wells, flat bottom) to test the OD600 and fluorescence intensity.
Measurement of fluorescence and OD were performed using the Thermo Scientific Varioskan Flash with the 'Thermo Scientific SkanIt' software and with the 96-Well Multiwell Plates (Corning).
And the measurement software we used is listed below: cited from http://www.thermo.com.cn/Resources/200802/productPDF_30632.pdf

Photometry

Fluorometry

Material and Equipment

BBa_K823005+BBa_I13504
BBa_K823008+BBa_I13504
BBa_K823013+BBa_I13504
Dh5a Ecoli cell
Atibiotics(Sigma)
LB culture medium
Corning 96-Well Multiwell Plates
Thermo Scientific Varioskan Flash

We use these parts of BioBricks, J23101+I13504, J23106+I13504 and J23117+I13504.There is only one difference between three, that they have different promoter.

Experimental Design

BBa_K823005+BBa_I13504:
We used 100mL Luria Broth Liquid media(102μg/mL chloramphenicol) in 250 mL conical flask to grow our cells(37℃，110rmp). Every 1h, we took 200μL in 96-well plate (round wells, flat bottom) to test the OD 600 and fluorescence intensity. We set our measure sample as triplicate per samples at a time units.
BBa_K823008+BBa_I13504：
The same design as the BBa_K823005+BBa_I13504.
BBa_K823013+BBa_I13504：
The same design as the BBa_K823005+BBa_I13504.

Expectation

Fluorescence and OD value were expected to increase in series containing BBa_K823005+BBa_I13504, BBa_K823008+BBa_I13504 and BBa_K823013+BBa_I13504 along with time, because the insert of Constitutive promoter. And the value of FLU/OD was expected to decrease.

Experimental Results

BBa_K823005+BBa_I13504（J23101+I13504）

BBa_K823008+BBa_I13504（J23106+I13504）

BBa_K823008+BBa_I13504（J23117+I13504）

Discussion

According to the data of BBa_K823005+BBa_I13504, we got the results adhering to our expectation among the OD value and the value of FLU/OD, but the fluorescence was increased at beginning and kept a balance, after 12 hour it began to decreased. We speculated the reason was the influence of bacteria density. According to the data of BBa_K823008+BBa_I13504, we got the results conformed to our expectation about the OD value and the fluorescence, but the value of FLU/OD decreased at beginning and increased after 8 hour. We speculated maybe the bacteria had stopped growing but the GFP was still increasing slowly. According to the data of BBa_K823008+BBa_I13504, the result of OD value, fluorescence and the value of FLU/OD were conformed to our expectation.

Team:BIT/Interlab Study