iGEM TecMonterrey team participates in the Interlab study

"The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. Can you measure fluorescence somewhere in your lab? Then this is the perfect study for you! Even if your lab or the organisms you work with mean that you can’t measure GFP from the specific devices, we want every team to be able to participate: email measurement at igem dot org and we will work out an alternative."

Eduardo Ramírez Montiel and Mercedes Vázquez Cantú were the two members in charge of the Interlab Study, and they did most of the actual work to acquire the measurements. Other people that should be credited are the remaining team members who also helped with this section. It should be noted that all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study.

The protocols began to be run on July 23th with the transformation of the biobricks. The next day a single colony from each of the selection plates was selected and inoculated in 20mL LB broth with the appropriate antibiotic. The tubes were incubated overnight at 37°C with 250rpm. Afterwards, a miniprep of the liquid cultures was performed and a glycerol stock was made.

On July 29th the restriction gels were run and all the parts purified except for the RFP. The next day another gel was made for the recuperation of RFP. On July 16th the ligations of D2-GFP, D3-GFP, D2-RFP and D3-RFP were done, and they were transformed the next day. However, no growth was observed in the selection plates. This protocol was repeated and finally on August 12th the D2-GFP ligation grew. The other ligations (D3-GFP [Amp] and D2-RFP [CAM]) were grown on September 4th. After this, the D3-GFP had to be cloned into a pSB1C3 vector but we weren’t able to do so before the deadline. Thus, a collaboration was made with the team UANL_Mty-Mexico. They gave us four D3-GFP ligations and we gave them a D2-GFP ligation.

At last, the ligations were confirmed on gel on September 16th. The measurements of fluorescence and optic density weren’t made until September 27th because the microplate lector was being used by another researcher.

Sample preparation

The following parts from the distribution kits were transformed in E. coli TOP10 cells according to the electrotransformation protocol:

• D1= 17F, plate 2, 2012 (Kn)

• D2= 20k, plate 1, 2013 (CAM)

• D3=20K, plate 1, 2012 (Amp)

• GFP= 12M, plate 1, 2012 (Amp)

• RFP= 9K, plate 1, 2014 (CAM)

After transformation, a single colony from each of the selection plates was selected and inoculated in 20mL LB broth with the appropriate antibiotic. These were incubated overnight at 37°C with 250rpm. Later, the tubes were centrifuged at 4,000rpm for 10 minutes and the pellet was resuspended in 1mL of LB media. Of the total volume, 425uL were used for a glycerol stock, which was stored at -80°C. The remaining volume was used to make minipreps. The DNA concentration and purity were measured with the nanodrop machine.

The digestion analysis of the parts was as following:

An electrophoresis gel was made to verify the length of the fragments. After the confirmation of the bands, they were purified by using the EZ-10 Spin column DNA gel extraction kit. The concentration of the isolated parts was measured with the nanodrop.

For the ligations, the calculations were made using the ligation calculator of the UT_Dallas 2011 iGEM team (https://2011.igem.org/Team:UT_Dallas/ligation). The ligations were incubated at 4°C for 16-24 hours. After the incubation, the ligation product was transformed following the electrotransformation protocol.

Again after transformation, a single colony from each of the selection plates was selected and inoculated in 20mL LB broth with appropriate antibiotic. The samples were incubated overnight at 37°C with 250rpm. Later, they were centrifuged at 4,000rpm for 10 minutes and the pellet was resuspended in 1mL of LB media. Of each sample, 425uL were used for a glycerol stock, which was stored a -80°C. The remaining 575uL were used to make minipreps. The DNA concentration and purity were measured with the nanodrop.

The digestion analysis of the parts was as following:

Stock colonies were inoculated in 20mL LB broth with appropriate antibiotic and grown overnight at 37°C with shaking (250 rpm). Afterwards, samples were inoculated in new LB broth with appropriate antibiotic and grown for 2 hours before the measurement of the optic density at 600nm and fluorescence (emission=360 and excitation=460 and 528). The measurements were done at 0, 4, 8, 12 and 16 hours.

For each sample, 3mL were centrifuged at 14,000rpm for one minute, and the pellet was washed twice with PBS buffer. Then, the pellet was resuspended in 600uL of PBS buffer, and 200uL of the sample were loaded in a 96-well Costar plate as follows:

Both green and red fluorescence were measured as well as optical density at 600nm. The control used for the OD₆₀₀ measurement was the blank LB medium without cells, and for the measurement assay the blank was TOP10 cells in PBS buffer. The measurements were taken every four hours for 16 hours. The time required for each measurement was about an hour and a half.

In order to determine whether to include or exclude each sample from the data set, the mean and standard deviation of the triplicates were calculated and with them the coefficient of variation was obtained. When this value was higher than 15, the atypical value was eliminated.

The main practical limit on the number and rate of measurements with this instruments and protocol is that the time required was too long; plus, there wasn’t enough available material to do many repetitions of each sample. No extra material was acquired for the Interlab Study; the only difficulty was to obtain permission to use the microplate reader.

The values of the measurement of the fluorescence and the optic density of the cultures can be found in the attached excel spreadsheet. The values are in triplicate and are reported for each four hours. Quantifications of other ligations are included in the spreadsheet, but only those that presented fluorescence and were confirmed by electrophoresis gel were reported.

OD_600NM vs Time

[Fluorescence (360/528) divided by OD_600NM] vs Time