Team:Brasil-USP/interlabstudy

Interlab Study

2nd International InterLab Measurement Study in synthetic biology


In order to characterize standard biological parts, fluorescence-based assays have been widely used with a variety of fluorophores such as fluorescent proteins (FPs). Green fluorescent protein (GFP) is frequently applied as reporter gene to confirm gene expression and analyse its regulation. The Second International InterLab Measurement Study in synthetic biology proposes to detect three promoters activity by measuring and comparing the levels of GFP expression of three devices expressing GFP and controlled by one of the three promoters (see Figure 1). The three promoters - BBa_J23101, BBa_J23106, BBa_J23117 - are from the Anderson library (http://parts.igem.org/Promoters/Catalog/Anderson), a constitutive promoter family with different strengths.1

Figure 1: Interlab devices 1, 2 and 3, in order from top to down.

Introduction and Motivation

In the eleventh iGEM edition happens the second Interlab Study. This study is based on the characterisation of standard biological parts and, as standard parts, it is fundamental to observe reproducibility and repeatability on their behaviour. Since all the teams have the same devices, we should confirm similar data values qualitative and hopefully quantitative for absolute units. Additionally, it will be possible to analyse the results to enlighten the differences between protocols. In this sense, Interlab study hopes to define well-characterised standard parts. We used a plate reader and a flow cytometer to measure fluorescence with biological and technical replicates, fulfilling InterLab study requirements and extra credit opportunity. Results showed mimimi

We understand the importance of this study and how it helps to define standards BioBricks and analyze the difference between labs and protocols.

Results

In the following subsections, we present our fluorescence measurements over time, a cytometry study showing a more microscopy view of how our cells behaved and our calculations of promoter strength using Relative Promoter Units (RPU). Notice that all of our data is publicly available here, and all software developed to calculate the results presented below are available here.

Fluorescence results - plate reader

As described above, we have recorded fluorescence and optical density (OD) over time of a 96-well plate organized as mentioned before. We show below fluorescence measurements after 11h of experiments, when all colonies presented an OD of about 0.5 u.a., except of course LB and LB+Cam which do not have any bacteria. Notice that we have for three biological replicates of three technical replicates, as suggested for extra credit. If you are interested in our complete data, we have made it public here.

Figure N (click for larger view): Fluorescence readings after 11h of experiment, not normalized by OD yet (see data below).

LB and LB+Cam are our baseline controls: they set the zero-level for the fluorescence. Although their levels are very close indeed, a Mann-Whitney U-statistic test showed that the antibiotic Cam is inducing a fluorescence that should not be disregarded (p-value < 0.5%). Thus, we will normalize all of our measures with respect to LB+Cam activity.

To better visualize this data, we show in the figure below The activity of each device averaged for each colony (considering the standard deviation). This fluorescence measure is normalized by LB+Cam. Notice how Device 3 fluorescence is close 1.0, i.e., not much is induced more than our zero-level activity. This is somehow expected, as this promoter is indeed very weak (see Relative Promoter Units below).

Cytometry

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Relative Promoter Units

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Measuring Promoter Strength using a Camera + Gimp

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Discussions

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Protocols

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Assemble protocol

All parts to assemble the devices were on pSB1C3 vector and were resuspended from iGEM kit 2015 in 10 μL of autoclaved Milli-Q water. 2.5 μL of these resuspended DNA were transformed in Escherichia coli DH5α cells and miniprepped to analyse bacterial clones. BBa_K823005, BBa_K823008 and BBa_K823013 (see table below) were digested with SpeI and PstI restriction enzymes (SP digestion), maintaining the promoters linked to pSB1C3 vector and then digestion products could be used in the ligation reactions (see protocols) with BBa_I13504, which was digested with XbaI and PstI (XP digestion). Three independent ligations were performed to obtain the required devices - K823005 + I13504 (Device 1), K823008 + I13504 (Device 2) and K823013 + I13504 (Device 3) - and final clones were confirmed with a digestion reaction with NotI enzyme (prefix and suffix). For restriction analysis, we selected two colonies of each device and separated population of DNA by agarose gel electrophoresis.

Figure 2: Restriction analysis of final clones using Gene Ruler 1Kb DNA Ladder (Thermo Scientific).

Sample preparation protocols

E. coli DH5α cells transformed with plasmids containing device 1, device 2 and device 3 were plated on LB agar (SIGMA) plates supplemented with chloramphenicol and grown for 18-20 hours at 37°C. Into independent sterile tubes with 5 ml of LB media (1:4) containing 34 μg mL-1 of chloramphenicol, three colonies of each device and control were tip from the LB agar plates and grown at 37 °C, 80 rpm overnight ( for 14-16 hours). All tests were performed in E. coli DH5α cells in high-copy plasmid pSB1C3.

Plate reader

Samples were prepared in a black, flat-bottomed, 96-well Costar plate by adding a volume of 200 µL per well of each sample (180 µL of LB media and 20 µL of pre-inoculum). We also measured optical density using a clear, round-bottomed, 96-well Greiner plate

Flow cytometer

Cultured bacteria were diluted to approximately 8 x 106 bacteria mL-1 or OD600 of ~ 0.01 (cell concentration must be in the range of 1x106 to 2 x 107 bacteria mL-1 according to the equipment manufacturer’s recommendation). At this concentration, we guarantee enough cells in the samples and avoid clusters formation. To reduce background fluorescence originated from components of the media, samples were washed twice with phosphate buffer saline (PBS) at centrifugation 5000 g for 5 minutes. The supernatant was removed and the pellet resuspended in 1 ml sheath fluid, same volume as used to obtain OD600 of 0.01.

Data acquisition protocol

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Plate reader

Data acquisition was performed in a plate reader model SpectraMax® M3 (Molecular Devices), SoftMax Pro software. To prevent cross-talk and light scattering, samples were prepared in a black plate and top read was set to acquire fluorescence measurements.

Flow cytometer

Flow cytometry measurements were acquired in a BD FACSCaliburTM Flow Cytometer (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA) equipped with BD FACSComp™ and BD CellQuest™ Pro software. First the equipment was calibrated to ensure optimal performance due to inherent instrument variability. As model calibrators we used BD Calibrite 3 Beads (BD Calibrite™) by running FACSComp program.

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