Team:Stony Brook/interlab

Interlab 2018

The 2018 Team participated in iGEM’s Fifth Interlab Study. This year’s interlab objective was to standardize fluorescence measurements by avoiding the use of optical density through a more direct method of counting colony-forming units (CFUs). This is because there is a lot of variability in synthetic biology based on different instruments, so this year’s study wanted to utilize a direct approach to measuring fluorescence. In order to conduct the study, the team transformed DH5-alpha Escherichia coli cells with eight plasmids that iGEM provided in the distribution kits. We followed iGEM’s transformation protocols for single tube transformation.

Calibration 1

First, we measured the OD600 of LUDOX CL-X in order to obtain a reference point. The results are pictured in Table 1.

Table 1: Calibration 1 Results

Calibration 2

Next, we conducted serial dilutions using silica beads that are the same size as E. coli to create a standard particle curve. As expected, the curve for the standard particles was linear. The results are pictured in graph 1 and graph 2.

Graph 1: Calibration 2 Particle Standard Curve

Graph 2: Calibration 2 Log-Scale Particle Standard Curve

Cell measurement protocol

After completing all of the calibration protocols, we transformed our E. coli with the two controls and the six test devices provided by iGEM and plated them onto chloramphenicol plates. After 16-18 hours of growth, we transferred two colonies from each plate into liquid culture with a total of 16 liquid cultures. We incubated these liquid cultures for 16-18 hours. After growing the cultures overnight, we made a 1:10 dilution and then measured the absorbance of the diluted cultures. Then, we furthered diluted them to a target absorbance of Abs600 = .2 for a final volume of 12 mL. We took out samples of these diluted cultures at time 0 and time 6. We measured the fluorescence and OD of the samples in a well plate. The data for absorbance and fluorescence are displayed in tables 2 through table 7.

Table 2: Abs600 Raw Readings

Table 3: Fluorescence Raw Readings

Table 4: Fluorescence per OD for time 0 (top) and time 6 (bottom)

Table 5: Net Absorbance at 600 for time 0 (top) and time 6 (bottom)

Table 6: Net fluorescein for time 0 (top) and time 6 (bottom)

Table 7: MEFL per particle

Colony Forming Units

In order to count the amount of cells per colony, we plated our positive and negative controls onto plates with chloramphenicol. We prepared our starting sample by diluting the positive and negative control colonies for an 8-fold dilution. After, we diluted the cultures further to a target OD of .1 and created triplicates of each culture for a final total of twelve. After, we conducted serial dilutions with the twelve samples. We plated dilution 3 (8x104) , dilution 4 (8 x 105) , and dilution 5 (8 x 106) on agar plates and incubated them overnight. After 18-20 hours, we counted the colonies on each of the plate and calculated the CFU/mL. The data is displayed in tables 8 through 10.

Table 8: OD of the starting samples

Table 9: Colony forming units per 1 mL of an OD600= .1 for positive colonies (BBa_I20270)

Table 10: Colony forming units per 1 mL of an OD600= .1 for negative colonies (Bba_R0040)