Team:UNITN-Trento/Measurement

InterLab Measurement Study

What happens when GFP meets different promoters? Are they all the same? Here is what we found out!

  • Introduction & Achievements

  • Experimental Design

  • Experiments& Protocols

  • Final Discussion & Results

The Interlab Measurement Study

The characterization of standard parts has always been one of the main concerns in Synthetic Biology. For this very same reason, iGEM teams from all around the World were suggested to take part in the biggest measurement study ever conducted and the 2015 UNITN-Trento iGEM team answered the call. The goal of this Second International Measurement Interlab Study is to assemble three different devices, each one containing a promoter with a screening plasmid intermediate and collect as many fluorescence data as possible. iGEM teams are free to use any technique to measure their devices as long the obtained data are solid and reproducible.

Clear Data and Protocols

We listed in the ”InterLab Study” page:

  • All devices measured for this study
  • All protocols developed and adopted
  • Sequencing data for all measurements devices

3 BioBrick devices

We used the three BioBrick devices listed in the ”Required Devices” section.

5 techniques

We used in-vivo and in-vitro techniques for measuring RNA and protein levels:

  • Plate Reader
  • Spectrofluorometer
  • Flow Cytometer
  • RT-qPCR
  • Cell-Free Extract

Statistical Reliability

We have three biological replicates for each measurement, with positive and negative controls

Worksheet and Protocol

We completed the InterLab Worksheet and the InterLab Protocol

Extra Credit Assignment

We did technical replicates for each sample

Experimental Design

We used the three mandatory devices for the measurement study:

The measurement devices were prepared by amplifying the reporter (BBa_I20270) by PCR. The amplified insert was then cut with XbaI and PstI and ligated into the plasmid containing the promoter previously cut with SpEI and PstI. All the devices were confirmed by restriction digestion as well as DNA sequencing.


In-vivo Measurements

The confirmed devices were then transformed in different bacterial strains of E. coli: NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorometer, and BD FACSCanto flow cytometer.


In-vitro Measurements

We also focused on transcription since the characterization is about promoters. To do so we performed RT-qPCR using a BioRad CFX96 Touch™ Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell-Free E. coli S30 Extract System with a Qiagen Rotor-Gene Q as the spectrofluorometer.

Experiments & Protocols

Extraction from the Registry

All the parts needed for the InterLab Study were extracted from the 2015 iGEM Registry Distribution Kit.

Part ID Description Plasmid Backbone 2015 Registry Location
BBa_K823005 Anderson promoter J23101 pSB1C3 Plate 1; 20K
BBa_K823008 Anderson promoter J23106 pSB1C3 Plate 1; 22A
BBa_K823013 Anderson promoter J23117 pSB1C3 Plate 1; 22K
BBa_I12504 RBS + GFP + 2 terminators pSB1A2 Plate 4; 21J
BBa_R0040 TetR sequence pSB1C3 Plate 2; 6F
BBa_I20270 Promoter MeasKit pSB1C3 Plate 3; 8P

Polymerase Chain Reaction (PCR)

Restriction Digestion of plasmids containing the promoter

Each promoter containing plasmid was digested with 1 μl of SpeI and 1 μl of PstI at 37°C overnight. The day after 1 μl of phosphatase (CIP from New England Biolabs) was added for 2 hours at 37°C. The enzymes were then heat deactivated.

The digestion reactions were assembled in this way:

PCR Products Plasmids
Template 3000 ng 2000 ng
Enzyme 1 2 μl 1.5 μl
Enzyme 2 2 μl 1.5 μl
Buffer (Stock 10X) 5 μl 5 μl
BSA (Stock 10X) 5 μl 5 μl
Water Up to 50 μl Up to 50 μl

Ligation

Plasmid:
Insert = 1:3		 Control
Vector 100 ng 100 ng
Insert 125 ng -
10X Buffer 2 μl 2 μl
Buffer (Stock 10X) 5 μl 5 μl
T4 - DNA Ligase 2 μl 2 μl
Water Up to 20 μl Up to 20 μl

The ligation reactions were assembled and incubated at room temperature for 1 hour according to the table beside. Subsequently 10 μl of the ligation mixture were transformed and plated into LB-agar plates with the proper antibiotic resistance. The confirmed devices were transfected in NEB10β, JM109, NEB Express.

Cells expressing the devices These three plates contains E. coli NEB Express transformed with J23101 (top left), J23106 (top right), and J23117 (bottom). Interestingly, promoters' strength are detectable by eye, depending on the colonies' color.

Cells expressing the devices. These three plates contains E. coli NEB Express transformed with J23101 (top left), J23106 (top right), and J23117 (bottom).

Parts Confirmation

Correct clones were screened by restriction digestion and confirmed by sequencing:

Cell pellets at the UV-trans illuminator Pellets of overnight NEB10β cells cultures expressing the assembled devices. Pellet's colors confirmed the promoter strength, (from left to right) the strongest promoter J23101, the medium strength promoter J23106, the weak promoter J23117, the positive control I20270, the negative control R0040.

Electrophoresis gel of all devices We digested three colonies for each device with XbaI and PstI. The correct assembly is confirmed by the band at 910 pb.

Glycerol stocks preparation and Sample Growth

A single colony was inoculated with a sterile pipette tip in a test tube with 10 ml of LB and antibiotic (1000:1 LB to antibiotic ratio) and placed in the thermoshaker (190 rpm, 37°C). When the culture got cloudy, 40 ml of LB+antibiotic were added to reach a final volume of 50 ml. The cells were grown until an OD600 of 0.5 and then centrifuged at 4100 rpm for 10 minutes at 4 °C. The supernatant was discarded and the cells were resuspend in 5 ml of LB + antibiotic + glycerol (20% v/v). The cells were kept on ice and were promptly aliquoted into 200 μl tubes and frozen at -80°C immediately. From this protocol we obtained a 10X concentrated glycerol stock for each sample.

The glycerol stock was thaw and added into 10 ml of LB with antibiotic, giving a starting culture with an OD600 of 0.1. The sample were grown in a 50 mL conical plastic tube in the termoshaker at 37°C and were grown until an OD600 0.7. At this point 3 ml of the culture were transferred in a new tube, centrifuged it, and stored at -20°C, unless otherwise indicated.

Fluorescence readings: Tecan INFINITE 200 PRO Plate Reader

Fluorescence readings: Cary Eclipse Fluorescence Spectrophotometer

Fluorescence readings: BioRad CFX96 Touch Real-Time PCR Detection System

The cells were grown from glycerol stock until an OD600 of 0.7 was reached. Total RNA was purified by using the Thermo Scientific GeneJET RNA Purification Kit, following the manufacturer`s instructions and subsequently genomic DNA was removed from the total RNA by using the Thermo Scientific RapidOut DNA Removal Kit, following the manufacturer`s instructions. RNA levels were quantified using NanoDrop 1000 and reverse transcription of cDNA from the RNA template was performed with Thermo Scientific RevertAid First Strand cDNA Synthesis Kit, following the manufacturer`s instructions. qPCR reactions were performed with BioRad CFX96 Touch Real-Time PCR Detection System (made in USA) and assembled as follows:

cDNA 5 ng
Primer Fw 180 ng
Primer Rv 180 ng
BioRad iQ SYBR® Green Supermix #1708880 Up to 10 μl
GFP Primer Fw ATGCTTTGCGAGATACCCAG
GFP Primer Rv TGTCTTGTAGTTCCCGTCATC
idnT Primer Fw CTGCCGTTGCGCTGTTTATT
idnT Primer Rv GATTTGCTCGATGGTGCGTC

Primers were designed for the reporter gene GFPmut3b and for the housekeeping gene idnT (D-gluconate transporter) as indicated above.

We then analyzed the raw data, calculating the relative fold expression of each GFP device compared to the housekeeping (ΔCt) and the related standard deviation:

Device Relative Fold Expression (A.U.) Standard Deviation
J23101 67,421.53 16,016.27
J23106 18,506.44 3,146.38
J23117 658.08 79.09
R0040 0.04 0.02
I20270 17,800.56 1,030.40

Fluorescence readings: BD FACSCanto Flow Cytometer

The cells were grown from glycerol stocks as described above. Differently from before when they reached the OD of 0.7 were not frozen, but were used immediately to measure fluorescence intensity. An aliquot of 5 μl of cells was diluted in 900 μl of PBS. The instrument used was a BD FACSCanto (made in USA) set with the following parameters:

  • FSC gain: 525 V
  • SSC gain: 403 V
  • FITC gain: 510 V
  • Flow rate: LOW
  • Total number of events in P2: 10000

Those parameters allowed the instrument to process 900/1500 events per second. We analyzed the raw data and plot the means of three replicates with relative standard deviations.

Fluorescence spectra from flow cytometer analysis of the NEB10β strain The merge shown above represents the devices J23101 (white), J23106 (yellow), J23117 (blue), and the negative control R0040 (orange). Data were analyzed using Cyflogic 1.2.


Fluorescence readings: E. coli S30 Extract System for DNA Circular

Miniprepped DNA was purified and extracted through phenol/chloroform extraction followed by ethanol precipitation. Reactions were set following Promega E. coli S30 Extract System Technical Bulletin and were performed using Qiagen Rotor-Gene Q (made in USA). Parameters for this specific experiment were set as follows:

  • Green channel gain: 0.67
  • Green channel Excitation: 365±20 nm
  • Green channel Emission: 510±5 nm

We analyzed the raw data and plot the means of three replicates over time:

Final Discussion

Our characterization confirmed the relative strength of the promoters

J23101 is the strongest promoter among the three, showing high expression of GFP in all three strains, regardless of the technique used. J23106 is the medium promoter and J23117 the weakest.

Ratios across promoters are kept the same

J23101/J23106 fluorescence ratios ranged from 2.0 to 4.5, depending on the strain and the technique. Differently from the other two promoters, J23117 showed a very low GFP production, as it was not detectable by eye or using the trans-illuminator and showed little fluorescence with the three techniques used.

Different techniques lead to the same results, with different sensitivities

The best way to perform a characterization is to use various techniques. Throughout our experiments we saw that each instrument has a specific sensitivity, which alters the output data. The FACS happened to be the most accurate among all, due to its extremely high intrinsic sensitivity. The plate reader also showed a good accuracy while the fluorometer was not able to detect the weakest promoter from the background noise. The use of the qPCR machine as a spectrofluorometer also gave positive results.

In vitro conditions mimic the in vivo reality

Comparing the results obtained from the cell-free extract to the others, we discovered that the promoters behave the same when working in vitro or in living bacteria. Since testing constructs in vitro is much faster than in vivo, our results suggest that it may be wise to first screen parts and/or genetic circuitry in vitro. Then the activity of a smaller subset could be confirmed with in vivo measurements.

Looking at promoters from a different angle

In vitro characterization by qPCR allows for the quantification of promoter strength by measuring RNA transcript levels, rather than by looking at the concentrations of something one step removed, i.e. protein. This approach gives a a more direct measure of promoter strength.

Promoter strength across strains and techniques Values presented are the average of three technical replicates for each of the three biological samples (total of 9 replicates). Data normalized on the maximum expression value of each technique. Heatmap calculated with R Project.



Bacterial strain does matter

The three promoters behaved differently in the different bacterial strains used. The bacterial strain which gave the highest fluorescence was NEB10β, which showed significantly increased expression of protein in all cases when compared with JM109 and NEB Express. Clearly this discordance among strains must to be due to their different genotypes. A different bacterial proteome (e.g. presence/lack of specific proteases and/or chaperonins) may alter protein production, processing and folding, and thus fluorescence emission.

Strain influence on promoters efficiency Values presented are the average of three technical replicates for each of the three biological samples (total of 9 replicates). Data normalized on the the maximum expression value of each technique. Heatmap calculated with R Project.