Team:BostonU/Interlab

From 2014.igem.org

Latest revision as of 01:13, 18 October 2014

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

Interlab Study

Section I: Provenance and Release Who did the actual work to acquire these measurements? High school student, Ariela Esmurria, worked on the BioBricks provided to prepare them for testing. Ariela digested and ligated the promoter and E0240 parts together into the backbones provided for J23101 and J23115 (backbone BBa_J61002). Afterwards, iGEM team member, Yash Agarwal, digested the J23101-E0240 and J23115-E0240 parts out of the J61002 backbone and ligated them into the appropriate destination vector (pSB1C3). Team mentor, Traci Haddock helped with troubleshooting some cloning problems What other people should be credited for these measurements? Kathleen Lewis, Alan Pacheco, Evan Appleton, Douglas Densmore On what dates were the protocols run and the measurements taken? Preliminary Testing was done on September 22nd to confirm fluorescence in all required constructs (J23100+E0240, J23101+E0240, I0260 and J23115+E0240). This was repeated on September 24th and the data was analyzed using the TASBE tools. Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study? Yes.

Section II: Protocol What protocol did you use to prepare samples for measurement? We used our Flow Cytometry Workflow protocol (see attached Excel file). What sort of instrument did you use to acquire measurements? BD LSRFortessa SORP flow cytometer with a High Throughput Sampler attached and FACS Diva 7.0 software How is it configured for your measurements? This Fortessa has five lasers, of which we used two for our measurements. We used the 488 nm laser for our GFP measurements and the 561 nm laser for our RFP measurements (used in our controls). For the GFP measurements, we also used a FITC channel 530/30 filter while for the RFP measurements we used a PE-Texas Red channel 610/20 filter. What protocol did you use to take measurements? As described in the Flow Cytometer workflow linked in the answer to question 1, we diluted overnight cultures of samples in PBS. We first perform a 1:100 dilution and check the concentration on the machine. If cell activity is more 3000 events/ second or less than 1500, we dilute according based on judgement. What method is used to determine whether to include or exclude each sample from the data set? We observed the histograms that the Fortessa produced in real time to decide whether a sample should be included in the data set. If the peak fluorescence for the FITC-A channel was more than or equal to 50 arbitrary units based on our FACS settings, it was included. This number was chosen based on our negative control cells, which show a peak below 50 fluorescence units and thus are considered to have no fluorescence emission recorded. As a result of our criteria, no samples were excluded from our data set. What exactly were the controls that you used? We used the following constructs as our controls. All of them were assembled using MoClo and are thus, not in BioBricks. • Positive GFP: J04B2Gm_AE = J23104 – BCD2 – E0040 – B0015 • Positive RFP: J04B2Rm_AE= J23104 – BCD2 – E1010 – B0015 • Dual GFP – RFP: COXGR_AF (J04B2Gm – J04B2Rm) • Dual RFP – GFP: COXRG_AF (J04B2Rm – J04B2Gm) • Negative control: E. coli DH5α Pro cells with no plasmid transformed • Rainbow beads for MEFL calculation: Spherotech Calibration Particles, 8-peak rainbow particles (Catalog # RCP-30-5A) What quantities were measured? We measured red and green fluorescence. How much time did it take to acquire each set of measurements? It took from about 30 minutes to 1 hour to acquire measurements for one 96-well plate, which held one set of samples. How much does it cost to acquire a set of measurements? We estimate our costs to under 20 cents per sample What are the practical limits on the number or rate of measurements taken with this instrument and protocol? There is an upper limit to the fluorescence that the machine can record (3 x 105 RFU). Another practical limitation is the number of plates that can be setup accurately at once using our Flow Cytometer workflow. A larger number of deep-well plates has a greater chance of error than a smaller one

Section III: Measured Quantities Units What are the units of the measurement? We report our data in molecules of equivalent fluorescein (MEFL), which is a standard unit of measurement. What is the equivalent unit expressed as a combination of the seven SI base units? (http://en.wikipedia.org/wiki/SI_base_unit) MEFLs are a standard unit of measurement but are not one of the seven SI base units. Precision What is the range of possible measured values for this quantity, using your instrument as configured for these measurements? (e.g., a meter stick measures in the range of 0 to 1 meter) Our instrument can measure up to 3 x 10^5 RFU. What are the significant figures for these measurement? At our highest reading, the measured data is to 15 significant figures. Is the precision the same across the entire range? If not, how does it differ? We prepare samples in triplicate in order to plot error bars in our result plots. There also exists a range of positive measured quantity that differs by machine to machine. Accuracy When was the instrument last calibrated? The instrument was calibrated 20 minutes before the experiment was run. We run CST beads provided by the manufacturer of the machine (BD) using the standard protocol every time a measurement set is prepared. How was the instrument calibrated? We calibrate the LSRFortessa using the BD Cytometer Setup and Tracking Beads (BD Catalog # 655051) for FACS Diva 7.0.

Section IV: Measurements We measured the following samples: • J23101 = BBa_J23101 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 backbone (high copy 100-300 copies) • I20260= BBa_J23101 + BBa_E0240 (B0032-E0040-B0015) in the pSB3K3 backbone (medium copy 20-30 copies) • J23115= BBa_J23115 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 backbone (high copy 100-300 copies) We measured fluorescence per cell for 50,000 cells per sample using our BD LSRFortessa flow cytometer. We measured each sample in biological triplicates, for a total of 150,000 cells measured per device. For GFP, we recorded the fluorescence data by exciting the cells with the 488 nm laser and capturing emission data using the FITC channel filter (530/30 filter) Measurements were recorded as arbitrary fluorescence units and converted to a standard unit, molecules of equivalent fluorescein (MEFL), using the TASBE tools (https://synbiotools.bbn.com/). The controls used for this experiment are given in the attached Excel file. We ran a preliminary test on September 22, 2014. Experimental detail and measurement results for Interlab Data collected on September 24, 2014 To download the completed workflow spreadsheet, click here. We ran our BioBrick samples in triplicate on the LSRFortessa flow cytometer, collecting 50,000 cells per sample well. For triplicates, we set up biological triplicates were the culture containing the plasmid of interest was struck out on an LB agar plate for colony isolation. Three colonies per plate were selected and grown in separate wells in a 96-well round bottom plate for testing. Cells were grown in 200 µL liquid culture (LB broth with the appropriate antibiotic – kanamycin for I20260 and chloramphenicol for J23101 and J23115) for 8 hours at 300 rpm at 37°C. Cells were then sub-cultured into a new 96-well round bottom plate by inoculating 195 µL of fresh media with 5 µL of the 8 hour culture. Plates were incubated for 16 hours at 300 rpm at 37°C. Prior to running the samples through the flow cytometer, samples were diluted 1:100 into 1xPBS (Phosphate Buffered Saline) by adding 2 µL of overnight culture into 198 µL of sterile 1xPBS in a 96-well round bottom plate. Contact Team Advisor, Traci Haddock (thaddockATbu.edu) for all raw Flow Cytometer data (reported by the machine in arbitrary fluorescence units). Using the TASBE tools and our controls (see above), we were able to convert our arbitrary fluorescence units into MEFLs. We report our MEFL results below.

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