Team:Austin UTexas/Interlab Contribution
Interlab Study Procedure
Protocol:
1. Transform the genetic parts from the BioBrick 2015 kit into TOP10 E. coli cells. BioBrick assembly was used to engineer the InterLab study devices. Refer to the BioBrick website for more information on assembly procedure: http://parts.igem.org/Assembly:Standard_assembly.
2. Streak out an agar plate (LB/CAM) with the E. coli containing the device and any controls
- streak out one plate/device and control
- incubate plates overnight (18-20 hours) at 37°C
- “negative or background” Control: Cell background control (TOP10 cells)
- Negative Control: LB only control
Inoculate liquid culture with experimental devices (in triplicate) and controls in test tube in 10 ml LB/CAM media
Incubate liquid cultures for 16-18 hours at 300 rpm in 37°C
Plate Reader Measurement Protocol:
- Obtain initial OD600 measurement of O/N cultures
- Dilute the samples to an OD600 of 0.5
- calculate the dilution needed for each sample
- dilute each sample
- Re-measure your sample on OD600 to make sure it is at least within 5% of 0.5
Measure your samples
- add 80ul of each sample to the wells of a clear-bottomed 96 well plate
- The 96-well plate was inserted into an Infinite 200 PRO Microplate Reader
- Then, using the Tecan i-control the proper settings were selected and recorded.
- The Settings were: Excitation at 480 nm (9 nm width) and Emission at 525 nm (20 nm width) with optimal gain.
Calculate standard deviation and mean across triplicates of each device
Flow Cytometry Protocol:
(needs to be filled out)
Extra Credit:
Three technical replicates of the experiment were recorded where the measurements were repeated. The experimental samples were measured at least three times with both the plate reader and flow cytometer. Furthermore, we conducted three days of measurements to observe any possible genetic breaking of the devices to confirm our hypothesis that the strongest promoter will confer the most metabolic load on the organism, which can cause loss-of-function mutations within the plasmid.
Protocol for further measurement of biological triplicates and technical replicates over the course of three days:
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Day 0:
- Streak out frozen stock on plate
- Use the inoculation loop to streak out your cells onto LB/CAM plate.
- Incubate your plate at 37°C overnight for at least 20 hours.
Day 1:
- Pick 3 independent colonies and start three 5 ml O/N cultures in LB/CAM media.
- Grow overnight at 37°C and at 200-225RPM
Day 2:
- Start fresh 1:1000 dilution 5 ml cultures for each O/N culture.
- vortex the culture to generate an homogenous mixture
- Remove 250 µl from each O/N culture
- Use 5 µl of this for your fresh O/N culture
- Measure fluorescence intensity for your cultures with Flow Cytometer
- From the remaining culture in the Eppendorf, add 200 µl to a well within our 96 well plate.
Day 3:
- Repeat the procedure for Day 2
- Record the fluorescence readings and Flow Cytometer from the previous day