Difference between revisions of "Team:NTU-Singapore/Measurement"

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We also spotted the mutants on an LB agar plate to have a qualitative view of the GFP brightness. The culture is diluted to OD<sub>600</sub> = 0.4 then 3uL of the culture is spotted on to the agar. Brightness of these spots parallels the results in the above graph. For example, 7GA, 8AG and 9GA is the brightest among the mutations on their respective base pairs while 12AC and 11AC are the darkest. <div align="centre"><img class="" src="https://static.igem.org/mediawiki/2015/3/3a/Rbs-spots.jpeg" width="821.6px" height="550px"></div>
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We also spotted the mutants on an LB agar plate to have a qualitative view of the GFP brightness. The culture is diluted to OD<sub>600</sub> = 0.4 then 3uL of the culture is spotted on to the agar. Brightness of these spots parallels the results in the above graph. For example, 7GA, 8AG and 9GA is the brightest among the mutations on their respective base pairs while 12AC and 11AC are the darkest. <div align="centre"><img class="" src="https://static.igem.org/mediawiki/2015/3/3a/Rbs-spots.jpeg" width="821.6px" height="550px"></div>
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Revision as of 15:16, 18 September 2015

NTU SG iGEM 2015




Measurement

Fluorescence measurement

Material
  • LB
  • MQ water
  • Micro-centrifuge
  • 96-well microplate
  • 50ml Falcon Tube
  • Tecan Infinite m200 microplate reader
  • PBS solution
Procedures
  1. Inoculate strains for overnight culture in 5mL LB
  2. Measure the OD600 of the overnight culture with 200uL of sample
  3. Dilute OD600 to 0.05 in 5mL of LB in 15mL tube
  4. Incubate at 30C for 4 hours so that bacteria reaches mid-log phase
  5. Dilute OD600 to 0.05 in 10mL of LB in 50mL tube
  6. Take 400uL of samples from culture media at one hour intervals starting from T=1hr
  7. Centrifuge at 15000rpm for 3 minutes
  8. Discard supernatant and resuspend pellet with 400uL PBS
  9. Take 150uL of sample and load onto microplate
  10. Measure OD600 and fluorescence intensity


Tecan Infinite M200 Measurement

Material
Parameters
  1. Excitation Wavelength: 485nm
  2. Emission Wavelength: 520nm
  3. Gain: 40


Ribosomal Binding Site

Growth Curve

As the measurements are carried out in six batches, the growth of mutants of the same batch are similar but differed a little among batches. This implies that GFP expression does not have any significant effect on the bacteris's growth.

1AT denotes A of base pair 1 is changed to T, the same is applied for other notations of RBS mutants.