Team:Cork Ireland/CollaborationsTrial
Interlab Study
The work was carried out by Brandon Malone.
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.
All persons involved consent to the data being published in subsequent studies.
Short intro:
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 diferent promotors from the Anderson series of promotors combined with a GFP part as outlined in the interlab study overview page.
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.
Timeline:
22/06/15 – individual promotors and GFP taken from the distribution kit and transformed into dh5 alpha. 17 F -4th plate= J23101
19 p-4th plate- J23117 4th plate
J23106- 17 P 4th plate
I13504-17 C 3rd plate-pSB1C3
Transformations were repeated on 25/06/15 as no colonies were present.
Plate 1 – 20K –K823005- J23101 –Psb1C3
Plate 1 – 22A J23106
Plate 1- 22kJ23117-Psb1c3
Here J23101 & J23117 had colonies, overnights were grown up with 3 liquid cultures prepared for each one.
Transformation repeated for J23106 with more DNA being used on 26/06/15.
27/06/15 Plasmid preps were made of Promotors and GFP generating biobrick. The plasmid DNA concentrations were then recorded using nanodrop.
Plasmids were restricted using biobrick standard restriction digests and then ligated togethed following gel extraction.
Eco + Spe used for promotors & Xba 1 + PSt1 used for GFP generator.
6/7/15. Following successful ligations for devices 1 and 2, overnights were prepared from the plates where some of the colonies that grew showed green fluorescence.
However the ligations involving promotor J23117 were unsuccessful in that colonies formed but none of which showed green fluorescence. Colony PCR was done to investigate and results showed that some of the correct size plasmids were there but there was no green fluorescence.
The primers used were the VF2 & VR2 primers which are complementary to the regions of the prefix and suffix. Commercially available solis biodyne was used as a ready to go mastermix of Taq polymerase, buffer and dNTPS.
Now both 3A assembly and traditional restriction/ ligation were trialled simultaneously to ligate J23117 and I13504.Each method of assembly was done in triplicate to increase the likelihood of success.
Following this on the 9th July, one of the colonies that formed on the plates showed green fluorescence and was taken and grew overnight.
Plasmid preps were made of all overnights that were successful and these were subsequently diluted 1:1000 and retransformed whereby a greater proportion of the colonies showed green flourecenece. 3 biological replicates were taken from these and by the 14th July, fluorescence was ready to be measures using the plate reader. As controls, colonies without promotors but containing the GFP generator I13594 were grown overnight and also LB media inoculated with antibiotic was also left overnight at 37 with the rest of the samples.
Each of the biological replicates was tested three different times using the plate reader. The average and standard deviation of both technical replicates and biological replicates was obtained. This average was then divided by the OD at 600nm.
Digests were performed on all the devices to show that they were the correct size by restriction mapping.
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)
X uL dH2O
X uL Plasmid DNA
2 uL 10x buffer
1 uL (each) enzyme
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.
Lane 1 = ladder
Lane 2=undigested plasmid of J23101 + I13504 ligation
Lane 3= device 1 digested with ECoRI and PstI
Lane 4= device 2 digested with EcoRI & PstI
Lane 5= device 3 digested with EcoRI & PstI
Run using an 0.8 % gel
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.
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. All of the devices were in the PSB1C3 high copy number plasmid backbone in order to reduce the number of variables in the experiment.
The three devices that are required to be studied are:
1) Device 1 is a composite of promotor J23101 and a RBS, GFP generator and a terminator I13504
2) Device 2 is a composite of promotor J23106 and the part I13504
3) Device 3 is a composite of promotor J23117 and the part I13504
All three devices were cloned in to a PSB1C3 vector.
Plate reader manufacturer