Team:UNITN-Trento/Test

Interlab Study

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  • 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 Interlab Study ever conducted and the 2015 UNITN 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. The three different promoters will differently affect the GFP production and thating plasmid intermediate and collect as 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 have 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 proteins levels:

  • Plate Reader
  • Spectrofluorimeter
  • FACS
  • RT-qPCR
  • Cell-Free Extract

Statistical Reliability

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

Worksheet and Protocol

We have completed the InterLab Worksheet and the InterLab Protocol

Technical replicates

We have 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 spectrofluorimeter, and BD FACSCanto FACS.


In-vitro Measurements

We also focused of transcription since the characterization is about promoters. To do so we 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 Circular DNA Real Time PCR.

Experiments &anp; 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 add 1 μl of phosphatase (CIP from New England Biolabs) 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

The ligation reactions were assembled and incubated at room temperature for 1 hour according to the table below:

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

Subsequently 10 μl of the ligation mixture were transformed and plated into LB-agar plates with the proper antibiotic resistance.

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

The confirmed devices were transfected in NEB10β, JM109, NEB Express.

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.

Fluorescence readings:
Tecan INFINITE ® 200 PRO Plate Reader

Fluorescence readings:
Cary Eclipse Fluorescence Spectrophotometer

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

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

Final Discussion