Difference between revisions of "Team:Sydney Australia/Interlab"

Revision as of 12:56, 31 October 2017

Overview

Nowadays, research extends well beyond the laboratory. The advancement of technology and social media has allowed for easier communication between labs across the world. The sharing of data between groups allows for more rapid development of scientific research, as well as greater reliability. With this being the fourth annual Interlab study, with many participating teams around the world, the ability for iGEM HQ to improve the procedure for the measurement of GFP so that it is reliable and repeatable, is possible and can be achieved with greater ease! As part of the fourth Interlab study, we transformed six devices, which are pSB1C3 plasmids with various GFP fluorescein inserts, as well as a pSB1C3 positive and negative control into DH5α cells. Two colonies of each transformants were selected to be measured for fluorescence with four replicates for each colony for greater reliability. The Interlab study allowed us to use the lab’s fancy plate reader, which is able to shake cultures at 37oC while simultaneously measuring the OD600 and fluorescence at our selected time intervals which was every 15 minutes for 6 hours. This was a lot of data to go through, however, getting to try out the fancy machine made it worthwhile! Before measuring our samples in the plate reader, the fluorescein standards, which are of a known concentration (uM) were used to calibrate the plate reader and provide a standard curve which would be used in the analysis of our data.

Results

Figure 1: A standard curve produced by measuring the fluorescence a series of dilutions of Fluorescein at an excitation wavelength of 485nm and an emission wavelength of 530nm. The curve above is an average of four replicates of the standard curve measured. A relatively linear trend was observed, with an increase in Fluorescein concentration (µM) revealing an increase in fluorescence.

Figure 2: The absorbance of all 6 devices, positive control, negative control, and the LB blank shown as an average of each of the four replicated for the two colonies for each conditions. The errors bars are the standard deviation of the data. All conditions apart from the LB+Chlor blank and Device 1 showed a steep increase in absorbance over time.

Figure 3: The measured fluorescence of all six devices, positive control, negative control, and the LB blank shown as an average of each of the four replicated for the two colonies for each conditions. excitation wavelength of 485nm and an emission wavelength of 530nm. The errors bars are the standard deviation of the data. All conditions apart from the LB+Chlor blank and Device 6, and the negative control increased substantially over time.

Figure 4: The [Fluorescein]/OD600 for each of the devices. The data presented is an average of each of the four replicates of both colonies for each device and error bars presented are the standard deviation for each of the data sets. The general trend seen in all six devices was that the ratio of fluorescein (µM/OD600) decreased over time.

Observations

It took many attempts to transform the test devices into our competent DH5Α cells
There was generally more deviation between replicates when measuring higher levels of fluorescence
Rather than storing samples on ice, we loaded the cultures into the 96 well plate and left them incubating and shaking in the plate reading while measuring readings. We felt that this would produce results of greater accuracy.
Initially, the fluorescence being measured from the standard curve was too high for the plate reader to read. The plate reader settings had to be adjusted in order to give readings.