Calibration 1:​
OD_​600​ Reference point - LUDOX Protocol

This calibration is to allow us to obtain a conversion factor which allows us to transform our absorbance (Abs₆₀₀) data from the plate reader into a comparable OD₆₀₀ measurement. This conversion is necessary as measurements of absorbance are dependent on volume, and the path length of the light defined by the fluid in the wells of the plate reader is unfixed and can vary from well to well.

Below is the data we obtained:

Calibration 2:​
Particle Standard Curve - Microsphere Protocol

For the second calibration, we performed a series of dilutions for the monodisperse silica microsphere and measured their Abs₆₀₀ in the plate reader. A standard curve of particle concentration was also constructed to convert Abs₆₀₀ measurements to a cell number estimate.

The log scale of the standard curve alters the originally relatively constant slanted line into a more exponential curve.

Calibration 3:​
Fluorescence standard curve - Fluorescein Protocol

For the third calibration, we hope to create a standard fluorescence curve in order to enable different teams to compare their fluorescence outputs. Therefore, we will prepare a serial dilution of fluorescein in four replicates and measure its fluorescence in a 96 well plate in the plate reader. Once measured, we can construct a standard fluorescence curve and use it to convert our cell-based readings to a corresponding fluorescein concentration.

For the fluorescence standard curve, we used a gain setting of 50 and a filter that passes a light wavelength of 528 nm / 20 nm. We had an emission wavelength of 525 and an excitation wavelength of 488.

Cell Measurement

We used E. coli K-12 DH5-alpha for our cell measurements and maintained all the same plates, volumes, and settings in our calibration process to ensure that the measurements are valid.

Regarding the observations during the experiment, it should be noted that our LB medium had an innately low fluorescence intensity. Also, from the data, we observed that the Abs₆₀₀ growth for Device 4 was limited but it is still possible to test its fluorescence. Moreover, regarding the dilution of target Abs₆₀₀ of 0.02, we first measured the 1:8 dilution of the overnight cultures and using the equations provided in the protocol, we calculated the amounts of source and LB needed to obtain the target Abs₆₀₀ of 0.02.

Protocol:
Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures

For the CFU protocol, which is used to calibrate OD₆₀₀ to colony forming unit counts, we have 2 Positive Control cultures and 2 Negative Control cultures. Our goal is to get the colony forming units (CFU) per 1mL of an OD₆₀₀ = 0.1 culture.

Using the equation provided in the protocol, we calculated how much culture and media we should add to dilute the OD₆₀₀ of our overnight culture to 0.1 in 1mL of LB + Cam media.

We successfully calibrated our OD₆₀₀ to colony forming unit counts as we were fairly accurate in ensuring that the OD₆₀₀ values were close to 0.1, which we believed was the most crucial part of the CFU protocol. Moreover, we utilized the data from dilution 4 for the conversion because it has the best countable number which is between 50 to 200 colonies.

We were fairly successful in completing the Interlab project as it was accepted by the iGEM committee the first time we submitted it. It was really quite an enjoyable side project to perform and to practice our experimental techniques in the lab.