Team:Dalhousie/InterLab

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Interlab

Scientific discovery depends on having valid results. Without these valid results it becomes difficult to make any real conclusions or progress. One way that variability can arise between teams is through measurements, especially measurements that involve complicated machines and procedures. The InterLab study is a way to test how different measurements can be between teams while performing the same experiment. This year the InterLab study is looking at variability in fluorescence, by having teams across the world measure the same glowing protein. More specifically, each team will look at the same six promoters and look at their effect on green fluorescent protein (GFP).

Background

Our Experiment

• We will be performing the same experiment as teams across the world. We clone genes into six sets of plasmids, each with the same GFP protein but different promoter regions and ribosome binding sites.

What are promoters?

• Promoters are sections of DNA that control the expression of genes, meaning they decide if the protein is created or not. Some people find it useful to think of promoters as switches on the assembly line, they don’t build the protein, but they need to be present for synthesis to occur. Its also important to know that not all promoters are the same, they differ in the amount of expression or synthesis they cause.

Constructs

• This experiment tested the effects of three promoters and two ribosome-binding sequences (RBS). All of the constructs used were delivered and used in plasmid form and encoded for chloramphenicol resistance. In total there were six constructs. Each had one of the following promoters j23106, j23101 and j23117. Along with one promoter, each construct also had one of the two RBS ( b0034 or j364100). Every construct had the same GFP (e0040) and terminator sequence (b0012).

Device 1

Device 2

Device 3

Device 4

Device 5

Device 6

Experimental

• Along with the six experimental constructs, iGEM included two control constructs to verify the measurements recorded. A positive control used the same constant GFP and terminator sequence with a known promoter (j23151) and RBS (b00032). A negative control was also included to safeguard against contamination. This construct had a pTetR promoter with no downstream GFP included.

Positive Control

Negative Control

Plate Reader

This experiment depended on accurate measurements of fluorescence to assess promoter sequence and RBS performance. For these measurements a plate reader and 96-well plates were used. OD600 was used to normalize the fluorescence with respect to the amount of cells in each well. This method is more accurate since it accounts for variation in bacteria concentration per well.

Calibration
The OD600 of the device was calibrated using a LUDOX solution. In a 96 well plate 100μl of LUDOX was added to wells A1, B1, C1, D1. Beside those 100μl of type 1 filtered water was added into wells A2, B2, C2, D2. This data created a correction factor that will convert raw OD600 values into standard OD600 values.

A FITC standard curve was used to calibrate the fluorescence values. FITC from the iGEM kit was spun down and re-suspended in 1ml of PBS to form a 2xFITC solution. The FITC solution was further diluted to 1x then used to make a standard curve with four replicates. The standard curve was made by starting with 200μl in well A1 with eleven wells of water to the right. After setting up the wells 100μl of FITC was transferred from well A1 into A2, diluting the neighboring well by a half. The following wells are diluted consecutively stopping at the final well making sure the final well is pure water. After transforming the constructs into competent E. coli the bacteria were plated on LB agar plates with chloramphenicol. After a day of growth the two colonies were chosen from each plate for inoculation in 10ml of LB media and chloramphenicol. The cultures were incubated on a shaker for 18 hours at 220 rpm at 37 degrees Celsius. On the third day the cells were diluted to a target OD600 and placed back into the incubator with the same conditions as before. Over next six hours 500μl four replicates of all cultures were taken (at times 0, 2, 4, 6) and put on ice.



After transforming the constructs into competent E. coli the bacteria were plated on LB agar plates with chloramphenicol. After a day of growth the two colonies where chosen from each plate for inoculation in 10ml of LB media and chloramphenicol. The cultures were incubated on a shaker for 18 hours at 220 rpm at 37 degrees Celsius. On the third day the cells were diluted to a target OD600 and placed back into the incubator with the same conditions as before. Over next six hours 500μl four replicates of all cultures were taken (at times 0, 2, 4, 6) and put on ice.

Results and Discussion

In both colonies 1 and 2, test devices containing the b0034 promoter showed higher fluorescence values than colonies with the j364100 promoter. In terms of the ribosome binding sites, the order in which they ranked was consistent between colonies and with different promoters. The most effective ribosome-binding site was j23101, followed by j23106 and then j23117.