Difference between revisions of "Team:Exeter/Interlab"

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InterLab

Introduction and Aims Protocol and Results

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

InterLab study began by measuring the standard LUDOX Abs600. Pipetted LUDOX and water into two separate columns of a 96 well plate and measured the absorbance of the 4 replicates at 600 nm on the standard Tecan mode.

A serial dilution of FITC provided in the interkab kit was prepared using PBS. Fluorescence of all samples was measured in standard measurement modes. Measurements were repeated to produce a series of standard curves. The fluorescence readings were taken at 26.4°C at 477 nm to 515 nm excitation and emission respectively. Gain setting measured at 46, 56, 66. A setting change of 10 units makes a significant difference to the fluorescence data. The plate reader was run at optimum gain setting. This set gain to 76, which was too high for these measurements as the weakest dilution had a lower fluorescence than the pure PBS. Ran the reader at 37°C and 56 gain to produce our optimal standard curve.

Fig. 1 Change in fluorescence for varied gain settings

Fig. 2 FITC standard curve of fluorescence

Competent cells of E.coli DH5α were prepared following the provided protocol. Tested the OD of the cells using standard settings (see iGEM 2016 file),aiiming for a reading of 0.4-0.5. The first reading produced an average of 0.2, after 15 minutes the average increased to 2.5. The cells were spun down and re-suspended in TF-1 and TF-2 buffer. 100µl was aliquoted into 1 ml Eppendorf tubes and 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) and added to LB media to make plates for the iGEM interlab parts transformed with different constructs.

The InterLab measurement kit contained 5 devices; (1) J23101+I13504, (2) J23106+I13504, (3) J23117+I13504, Positive and Negative control. The first attempt to transform 5µL of each device into our competent cells found no liquid in the tubes provided. The provided tubes were re-suspended using elution buffer from a Qiagen mini-prep kit. A transformation of plasmids used in the lab under a strong promoter and GFP was performed mirroring the Interlab protocol to determine if the cells were competent. Spread plates were made and incubated at 37°C.

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. A new interlab kit was ordered from iGEM.

New plasmid constructs arrived and positive and negative controls were obtained from the kit plate provided. Resuspended DNA from the iGEM registry plates in 10μl of MiliQ water. 5μl of each resuspension solution was used for the transformation protocol. Original competent cells served as an additional negative control. Transformation was completed and cells were plated onto onto 80μl/ml chloramphenicol plates.

All the colonies had grown, however the positive and negative controls had a weak fluorescence intensity. The positive control had the fewest colonies and lowest 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.

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 using the calibrated Tecan spectrometer.

The protocol was improved by using a latin rectangle arrangement in another 96 well plate. A lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. 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. Sterile LB was loaded into peripheral wells to lessen edge effects which can skew results due to high evaporation at the periphery. These adaptations aim to provide more accurate data by reducing plate effects, samples are spread randomly around the plate so as to minimise differences in results due to irregularities in plate temperature.

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 shaking incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator briefly to warm up.

Fig. 3

Fig. 4

Flow Cytometer

Part two of the InterLab study was started on the 31/8.Overnight cultures of the devices and controls were grown from glycerol stocks inside a 15ml falcon tube with 5ml of LB media and 5μl of chloramphenicol.

An initial OD600 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. Adjustments were made to the side-scatter and forward scatter PMT voltages using negative control bacteria to centre the distribution, adjustments to FITC/GFP PMT voltage were made using the positive control bacteria to align bell curve an order of magnitude below the upper end of the scale. 1,000 events were acquired from calibration beads and from each biological sample.

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. No problems were encountered using the protocol or FACS machine.

@@ Line 676: / Line 676: @@
 		<p id="pp">InterLab study began by measuring the standard LUDOX Abs600. Pipetted LUDOX and water into two separate columns of a 96 well plate and measured the absorbance of the 4 replicates at 600 nm on the standard Tecan mode.</p>
-<p id="pp">A serial dilution of FITC provided in the interkab kit was prepared using PBS. Fluorescence of all samples was measured in standard measurement modes. Measurements were repeated to produce a series of standard curves. The fluorescence readings were taken at 26.4°C at 477 nm to 515 nm excitation and emission respectively. Gain setting measured at 46,56,66. A setting change of 10 units makes a significant difference to the fluorescence data. The plate reader was run at optimum gain setting. This set gain to 76, which was too high for these measurements as the weakest dilution had a lower fluorescence than the pure PBS. Ran the reader at 37°C and 56 gain to produce our optimal standard curve.</p>
+<p id="pp">A serial dilution of FITC provided in the interkab kit was prepared using PBS. Fluorescence of all samples was measured in standard measurement modes. Measurements were repeated to produce a series of standard curves. The fluorescence readings were taken at 26.4°C at 477 nm to 515 nm excitation and emission respectively. Gain setting measured at 46, 56, 66. A setting change of 10 units makes a significant difference to the fluorescence data. The plate reader was run at optimum gain setting. This set gain to 76, which was too high for these measurements as the weakest dilution had a lower fluorescence than the pure PBS. Ran the reader at 37°C and 56 gain to produce our optimal standard curve.</p>
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