Difference between revisions of "Team:Lethbridge/InterLab"

Overview

The ability for parts to provide repeatable outputs is key to engineering applications, including synthetic biology. The purpose of the 2017 interlab study was to address how reproducible and at what levels protein expression could be driven by standardized promoters and ribosomal binding sites. Constructs were provided by the iGEM measurement committee that utilized green fluorescent protein as a reporter. This allowed interlab study contributors to easily follow protein expression driven by the standardized promoters and ribosomal binding sites.

In order to better compare expression levels between research groups, the 2017 interlab study incorporated calibration protocols to allow for reporting of fluorescence levels in absolute levels. Specifically, fluorescence levels from biological samples were calibrated against fluorescence from a fluorescein standard curve, allowing for fluorescence results to be reported in terms of the amount of fluorescence produced by a certain concentration of fluorescein.

Test Devices

The negative control for the interlab study did not contain DNA coding for recombinant fluorescent-protein expression , thus was well-suited for examining background fluorescence of cellular components. The remaining constructs contained GFP (E0040) under the control of various promoters and ribosomal binding sites. The promoters and ribosomal binding sites utilized for Test Devices 1-6 are outlined below. All constructs contained E0040, B0010, and B0012 following the promoter and ribosomal binding site.

Methods

All methods were based on the iGEM InterLab 2017 Plate Reader Protocol found here:


Calibration
The calibration protocol for the OD 600 point was performed, followed by the generation of a fluorescein fluorescence standard curve. More details of calibration protocols can be found here:

Day 1
Following the iGEM InterLab protocol, the eight plasmids, Positive Control, Negative Control and Test Device 1-6, were transformed into E. coli DH5-alpha cells.

Day 2
Two colonies were picked from each plate and grown overnight in 5 mL of LB + Chloramphenicol at 37 ºC and 220 rpm.

Day 3
According to the provided interlab study protocol, OD₆₀₀ and fluorescence were measured (excitation 485 nm, emission 530 nm) by our instrument at 0, 2, 4 and 6 hours. Full details of the protocol can be found here:

Results

Calibration: Fluorescein Standard Curve
The instrument utilized for our data displayed a fluorescein-dependent increase in fluorescence. Data was plotted with both a linear, and a log scale x-axis (Figure 1). At very high fluorescein concentrations (25 and 50 𝛍M) the data displayed a non-linear trend when plotted with a linear x-axis (Figure 1A), likely due to over saturation of the detector. However, the remaining details of the calibration curves are consistent with the result desired by the iGEM interlab committee (values forming a straight line on linear and log scale x-axis, 1:1 slope, etc.)

Figure 1 - Fluorescein Calibration Curve. A Linear B Log scale Expression of Fluorescent Proteins from Test Devices
Cell growth and fluorescent protein expression from the six test devices and the two controls displayed varying levels of 𝛍M Fluorescein/ OD₆₀₀ (Figure 2). As expected, the negative control construct exhibited little background fluorescence for both biological replicates. The positive control construct exhibited reproducible patterns of fluorescent protein expression over the course of the experiment, with 𝛍M Fluorescein/ OD600 peaking at the two hour time points for both biological replicates. Test Device 1 exhibited variable, but high 𝛍M Fluorescein/ OD₆₀₀ values throughout the course of the experiment. While Test Device 2 exhibited mid-range levels of 𝛍M Fluorescein/ OD₆₀₀ values, peaking at the two hour time point. Test Device 3 exhibited background levels of 𝛍M Fluorescein/ OD₆₀₀ throughout the course of the experiment. And Test Device 4 displayed high-levels of 𝛍M Fluorescein/ OD₆₀₀, with the highest expression levels occurring from 2 hours onward. Test Device 5 displayed low, but above background levels of fluorescence with 𝛍M Fluorescein/ OD₆₀₀ peaking at the 2 hour time point. Whereas Test Device 6 displayed background levels of 𝛍M Fluorescein/ OD₆₀₀.

Figure 2 - Fluorescent Protein Expression of Test Devices. Fluorescent and OD₆₀₀ measurements obtained at 0, 2, 4 and 6 hours represented as a bar graph in 𝛍M Fluorescein/ OD₆₀₀ for the two controls and 6 test devices.

Promoter Influence
The lowest levels of fluorescent protein expression came from Test Devices 3 and 6. These devices both utilized the J23117 promoter, which had been previously characterized as being the weakest-strength of the promoters tested in the 2017 interlab study (see J23100-J23119 on the registry of standard biological parts: http://parts.igem.org/Part:BBa_J23117). In terms of the remaining two promoters, constructs utilizing J23101 displayed higher fluorescence levels than constructs utilizing J23106. Because of this, we claim the relative strength of the promoters is J23101>J23106>J23117.

RBS Influence
When comparing Test Device 2 to Test Device 5, it is clear that the B0034 ribosomal binding site utilized by Test Device 2 elicits higher levels of protein expression than the J634100 ribosomal binding site utilized by Test Device 5. As both devices contained the same promoter, differences in protein expression levels are attributed to differences in the ribosomal binding site. This trend of B0034 driving higher protein expression that J634100 continues when comparing Test Devices 1 and 3, where Test Device 1 contains B0034. Because of this, we therefore claim that B0034 drives protein expression at higher levels than J634100. In terms of absolute expression values (in terms of 𝛍M Fluorescein/ OD600) our results will be compiled with teams who completed the interlab study, and analyze how reproducible these absolute expression levels are among labs worldwide.