Introduction and Aims
Interlab study for 2016 iGEM aimed to quantify expression of five different
reporter constructs which have GFP under the control of differing promoters and ribosome
binding sequences. This involved measuring the fluorescence/OD600 of the total population
of DH5α E.coli cells with each construct to give an adjustment for relative expression per cell.
The objective is to convert the relative unit of fluorescence to an absolute unit by calibrating
each team’s instruments and comparing this to a measured standard curve.
Protocol and Results
Our team began the InterLab study on day 1 by measuring the standard LUDOX Abs600. This involved pipetting LUDOX and water into two separate columns of our 96 well plate and measuring the absorbance of the 4 replicates at 600 nm on the standard Tecan mode.
Later the same day we prepared a serial dilution of FITC provided in the interkab kit using PBS. This was pipetted into a 96 well plate and the fluorescence of all samples were measured in standard measurement modes. We then repeated these measurements to produce a series of standard curves. The fluorescence readings were taken at around 26.4°C at 477nm to 515nm excitation and emission respectively. We measured at a gain setting of 46,56,66. We found that 10 makes a significant difference to the fluorescence data. We then ran the plate reader on the optimum gain setting and then set the readers gain to 76. This was too high in our opinion as the weakest dilution had a lower fluorescence than the pure PBS. We then ran the reader at 37°C and 56 gain to produce our optimal standard curve.
The next day in the lab we finished preparing competent cells of E.coli DH5α following the provided protocol.Firstly we tested the OD of the cells using our standard settings (see iGEM 2016 file), we were aiming for a reading of 0.4-0.5. The reading came out at an average of 0.2. We waited another 15 minutes as they should have been growing quickly. We took another OD reading and it had increase to an average of 2.5. It was decided that the reader was giving a strange measurement and we continued with the protocol.The cells were spun down and re-suspended in TF-1 and TF-2 buffer. 100µl was aliquoted into 1 ml Eppendorf tubes and these were immediately dropped into liquid nitrogen. The cells were then left in the -80°C freezer. Chloramphenicol was made as a stock of 25mg/ml (weight measured was 76.6mg). This was added to the LB media we were going to make our plates for the iGEM interlab parts. We were now ready to start the transformation of the different constructs.
The InterLab measurement kit contained 5 devices; (1) J23101+I13504, (2) J23106+I13504, (3) J23117+I13504, Positive and Negative control. Upon removing these from our freezer and attempting to transform 5µL of each device into our competent cells we found we had no liquid in the tubes provided. We then re-suspended the provided tubes using elution buffer from a Qiagen mini-prep kit. A transformation of plasmids that had already been used in the lab under a strong promoter and GFP was performed mirroring the protocol we had carried out when attempting to transform the InterLab constructs. This was done in order to determine if the cells were competent. Spread plates were conducted and all the plates were incubated at 37°C.
We checked our plates the next day to find that the competent cells containing the previously used strong promoter and GFP coding sequence showed strong expression of GFP. However the competent cells transformed with the iGEM parts showed no growth and so a new interlab kit was ordered from iGEM.
New plasmid constructs arrived and we were instructed to obtain positive and negative controls from the kit plate provided. We resuspended DNA from the iGEM registry plates in 10μl of MiliQ water. 5μl of each resuspension solution was used for the transformation protocol. We left one batch of competent cells untouched to serve as an additional negative control. After the transformation was completed the cells were plated onto onto 80μl/ml chloramphenicol plates.
All the colonies showed growth the next day. However the positive and negative controls had a weak fluorescence intensity. Curiously the positive control had the fewest colonies and fluorescence intensity. The ranking of strongest to weakest fluorescence intensity for the constructs followed the following order: P1, P2, P3. Consequently, 5ml overnights of all the successful transformations were produced and left to grow overnight in the 37℃, 220 rpm shaking incubator.
The next day involved cell growth, sampling and assaying. The cultures were removed from the incubator and 100μl of each overnight was pipetted into the 96 well plate in order to obtain their OD at the calibration setting.
| Contruct |
Starting OD |
| P1 |
0.701 |
| P2 |
0.614 |
| P3 |
0.615 |
| Positive |
0.587 |
| Negative |
0.593 |
The cultures were diluted to achieve an OD600 of 0.02 and incubated at 37℃, 220 rpm .
Measurements were then taken each hour for 6 hours by pipetting 100µl of each culture into the 96 well plate according to the iGEM layout for Abs600 and Fluorescence measurement and placing this into the calibrated Tecan spectrometer.
In addition to this, our team decided to improve the protocol by using a latin rectangle arrangement in another 96 well plate. Our lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini vibrating incubator at 37°C each time in between plate readings. A scratch was noticed on the plate lid so we replaced it with a different lid. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator for 1.46 mins to warm up.
The Latin rectangle is designed so that no sample gets pipetted more than once in any row or column. The order in which the samples get arranged was worked out using the random number function of Microsoft Excel. We also loaded sterile LB along the outside of the 96 well plate in order to lessen the effects caused by edge effects.
Edge effects can skew results. Liquid from samples in the outer wells is more likely to evaporate than liquid from samples closer to the middle. This creates condensation as well as lessening the amount of sample found in some of the wells, which can influence the outcome of the measurements.
Through these adaptations we believe we have provided more accurate data.
Flow Cytometer
We started the second part of the InterLab study on the 31/8. Glycerol stocks of the cells containing the studied constructs were scratched with a pipette tip in order to prepare triplicate cell cultures. The cultures were incubated overnight inside a 15ml falcon tube with 5ml of LB media and 5μl of chloramphenicol.
The next day all cells had successfully grown. An initial OD reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator.
The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website.
Our results along with the completed worksheet were submitted on the 1/9. We didn’t encounter any problems with the protocol nor using the FACS machine.
