Team:Cork Ireland/CollaborationsTrial
Interlab Study
The purpose of the interlab study is to measure fluorescence data from three devices. iGEM teams from around the world are invited to take part in this study and analyse the difference in fluorescence from three devices i.e different promotors from the Anderson series of promotors combined with a GFP part as outlined in the interlab study overview page. We transformed the three devices once they were constructed into E.coli and then measured our results using a plate reader.
Devices
The three devices that are required to be studied are:
- Device 1 is a composite of promotor J23101 and a RBS, GFP generator and a terminator I13504
- Device 2 is a composite of promotor J23106 and the part I13504
- Device 3 is a composite of promotor J23117 and the part I13504
All three devices were cloned in to a PSB1C3 vector.
Both positive and negative controls were also used, the part BBa_I20260 prepared from the distribution kit was used as the positive control. This contains the promotor J23101 with the GFP generating device but was placed into a PSB3K3 plasmid backbone. For the negative controls, the plasmid with the part BBa_I13504 or GFP generating device but no promoter was grown in a liquid culture overnight. LB media inoculated with Chloramphenicol was also left to shake with the other samples overnight and subsequently its fluorescence measured.
Assembly
The promotor’s respective names are J23101, J23106 & J23117. The GFP part is I13504. Each part is present in the distribution kit, the first step of the project was therefore to assemble the devices (promotor plus GFP generator). Due to difficulties encountered experimentally more than one assembly method was trialled. 3A assembly was carried out and so too was the traditional restrict & ligation method.
Provenance and releases:
Who did the work for the interlab study?
The work was carried out by Brandon Malone.
When was the work carried out for the study?
The devices were constructed over a period from June 26th to July 17th. The measurements were done at the start of the following week and the data was analysed and graphed shortly afterwards.
Do you consent to your data being included in subsequent publications?
All persons involved consent to the data being published in subsequent studies.
What is model of measuring tool & how is it configured?
The devices were measured using a plate reader. The plate reader used was the Tecan infinite 200 with tecan-i-control software. The plate reader was used to get the absorbance at 600nm and subsequently the fluorescent read-out was got by exciting the cells with a wavelength of 485 nm and capturing the emitted wavelength with a channel of 532nm. The instrument uses a xenon-flash lamp as a light source.
Materials used and protocols:
Devices
All devices made were transformed into DH5 alpha cells. As outlined previously the 3 devices to be studied were made using either of 2 cloning methods, 3A assembly or the more traditional restriction digest/ Ligation method. The positive control BBa_I20260 was taken from the spring distribution kit and grown up in triplicate, the negative control BBa_I13504 alone was also taken from the spring distribution kit and grown up. And of course the respective promoters were taken from the spring distribution kit transformed and grown as liquid cultures. Plasmid minipreps were subsequently done to isolate purified plasmids which were digested to allow for cloning.
Cloning Protocol-traditional restriction & ligation
The following applies to each of the three devices. The promotor parts shall be from here on known as the “promotor” and the part BBa_I13504 shall be known as GFP generator.
After plasmid prep, restrict both parts for the device. Digest 1 microgram approx. of Promotor DNA with EcoRI and SpeI and roughly 1 microgram of GFP generator DNA with XbaI and PstI.
A typical protocol is as follows:
Pipette the following into a PCR tube and incubate for the required amount of time (normally between 1 hr and 6 hrs depending on enzyme activity, amount of enzyme used and DNA concentration)
Following this the digests should be run on an agarose gel. The bands containing the required parts should be cut and using a commercially available Gel extraction kit or the correct reagents, the DNA should be extracted from the gel. DNA concentration should be obtained using a nanodrop.
Following this the parts can be cloned together using DNA ligase. The result of the cloning should be transformed into LB media with the correct antibiotic resistance.
Note: The above method worked for device 1 & 2, 3A assembly was trialed for device 3 following cloning difficulties. The devices were checked if the correct insert was present by doing a digest using the restriction enzymes EcoRI and PstI. Following successful ligations, the resultant fluorescent colonies were picked in triplicate and grown in a liquid cultures overnight so that subsequently on the following day the fluorescence could be measured using a plate reader.
Plate reader protocol:
In order to measure the fluorescence, liquid cultures were grown of all the biological replicates. 100uL of these cultures was then pipetted into a 96 well plate and the OD 600 was measured. Dilutions were made of the absorbance at 600nm to give an OD 600 close to 0.5 in order to normalise the results across all the replicates. 100uL of the overnight was pipetted out 2 more times and diluted in order to provide technical replicates. Technical replicates all were derived from the same colony as the initial biological replicate.
The samples on the 96 well plate were then analysed. The OD600 was measured and the fluorescence was measured using 488 nm wavelength to excite the sample and 532 nm wavelength for emission.
Results and discussion
The results obtained showed for the most part what would have been expected, the promotor J23101 and GFP generator had the highest fluorescence. The understanding from previous work is that J23101 is the strongest promotor, J23106 the next one after that and the weakest being J23117. The results of the experimentation somewhat supported this view although the relative strengths of the promotors were slightly different from what was previously recorded. The positive control which was the same parts as device 1 i.e J23101 plus the GFP generating part but in the medium copy number plasmid PSB3K3 was weaker than the same device in the PSB1C3 plasmid. All of the devices were in the PSB1C3 high copy number plasmid backbone in order to reduce the number of variables in the experiment apart from that of the negative control. As can be seen in the data, there was one outlier present for Device 2, the fluorescence measured for the biological replicate 3 was well below the other two but due to the relatively small sample size, we chose not to exclude it from the data set.
