iGEM 2018 — Team William_and_Mary

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# Team:William and Mary/Notebook

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[![](https://static.igem.org/mediawiki/2018/e/ec/T--William_and_Mary--DecodeLogo1.jpg)](https://2018.igem.org/Team%3AWilliam_and_Mary)

* [Project](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Background](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Results](https://2018.igem.org/Team%3AWilliam_and_Mary/Results)
* [Temperature Systems](https://2018.igem.org/Team%3AWilliam_and_Mary/Heat)
* [Methods and Data](https://2018.igem.org/Team%3AWilliam_and_Mary/Experiments)

- [3G Assembly](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Method Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Collaborations](https://2018.igem.org/Team%3AWilliam_and_Mary/Collaborations)
- [Parallel Circuit Testing](https://2018.igem.org/Team%3AWilliam_and_Mary/3G_Mixed)
- [3G Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
- [Protocol](https://2018.igem.org/Team%3AWilliam_and_Mary/3GProtocols)
- [Modeling](https://2018.igem.org/Team%3AWilliam_and_Mary/Model)
- [Human Practices](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Incorporating New Communities](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Incorporating_new_communities)
  * [A Statewide Standard](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/A_Statewide_Standard)
  * [Continuing the Conversation](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Continuing_the_Conversation)
  * [IHP/Silver](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices)
- Judging
  * [Medal Requirements](https://2018.igem.org/Team%3AWilliam_and_Mary/Medal%20Requirements)
  * [Measurement](https://2018.igem.org/Team%3AWilliam_and_Mary/Measurement)
  * [Demonstrate](https://2018.igem.org/Team%3AWilliam_and_Mary/Demonstrate)
  * [Best Part Collection](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
  * [Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Parts)
  * [Improve](https://2018.igem.org/Team%3AWilliam_and_Mary/Improve)
  * [Interlab](https://2018.igem.org/Team%3AWilliam_and_Mary/InterLab)
- [Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Meet the Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Attributions](https://2018.igem.org/Team%3AWilliam_and_Mary/Attributions)
  * [Notebook](https://2018.igem.org/Team%3AWilliam_and_Mary/Notebook)
  * [Protocols](https://2018.igem.org/Team%3AWilliam_and_Mary/Protocols)
  * [Safety](https://2018.igem.org/Team%3AWilliam_and_Mary/Safety)

# Notebook

**Week 1: 180528 -180604**

* Began cloning 3G parts based off the library sent from the Murray lab
* Researched literature on dual inducible promoters
* Sent out several cloned 3G parts for sequencing
* Planned out several IFFLs to test.

**Week 2: 180604 -180610**

* Continued cloning 3G parts onto our standard WM Pad backbone
* Redesigned primers for cloning 3G promoter parts, as previous ones were faulty
* Received results from sequencing, confirmed parts
* Beginning to assemble circuits using 3G assembly
* Making inducible reporter construct and an mf-Lon based IFFL

**Week 3: 180611 -180617**

* Completed cloning the portion of our comprehensive 3G assembly part library needed for our circuits
* Adding remaining 3G parts from Murray’s lab to our library
* Working on designing and building a dual inducible promoter and low copy backbone for assembly
* Confirmed minipreps based on sequencing results
* Started plate reader for experiments on circuits with IPTG and ATC inducer
* Started using FACS machine for experiments

**Week 4: 180618 - 180624**

* Microscopy training with Jacob Sawyer, advanced imaging specialist from Nikon
* Spin down inducer tests to see if we can remove inducer from media to create pulses
* Plate reader experiment of pulse timing in different conditions
* FACs experiment of how long cells can be incubated on ice without changing results
* Decided to stop sequencing 3G parts and start functionally screening them instead
* Set up our first Golden Gate reaction
* Madilyn & Friends Outreach event

**Week 5: 180625 -180701**

* Continue designing and building circuits
* Continue inducer spin down tests for plate reader experiment
* Reached out to St. Andrews to solidify collaboration partnership
* Considering alternatives for the inducer spin down tests due to multiple complications
* Looked into light-induced gene expression systems
* SEP 8th graders

**Week 6: 180702 -180708**

* Experienced some leakiness with our FACS machine, spent time fixing this
* Abandoned light-induced gene expression system, as it appears impractical in lab and less relevant to in vivo systems
* Began looking into temperature-induced circuits
* Continue inducer spin down tests
* Making different variants of the same circuit with different strength repressors
* Made and characterized Oscillator Circuit
* Cell sorting with GS, image dissociated cells on the slides
* Started Interlab Study measurements

**Week 7: 180709 -180715**

* Successfully cloned our entire 3G library, except recently added parts
* Demonstrated cells can be frozen in 15% glycerol without changing results
* Successfully tested whether inducers can be removed from media
* Using parts from last year, tested an IFFL with a single 20 minute pulse
* Tested an IFFL with a single 40 minute pulse
* Began assembling and testing circuits utilizing temperature as an inducer (TS-C1 system)
* Attended the Mid-Atlantic Meetup hosted by University of Maryland
* Submitted Interlab measurements

**Week 8: 180716 -180722**

* Reached out to UVA for collaboration
* Continued with plate reader experiments
* Continued testing heat inducible circuits
* Cloned and transformed Oscillator parts for functional testing
* Medical Explorers Outreach Event Visit Day 1 and 2
* Building with Biology event
* Camp Launch

**Week 9: 180723 -180729**

* Contacted University of Pittsburgh for collaboration
* Continued plate reader experiments
* Testing lower temperature inductions
* Successfully created working high copy inducible parts and possible working low copy parts
* Addressing issues with microscopy
* Plus S Ice Cream and iGEM

**Week 10: 180730 -180805**

* Continuing plate reader experiments
* Interlab data got rejected due to accidentally switching the Od600 and fluorescence in the form. Minor issue, easily resolved and submitted
* Investigated parameters for oscillator
* Sent collaboration package to UVA and University of Pittsburgh
* Started considering clontegration and requisite parts and primers
* First MUN Meeting

**Week 11: 180827 -180902**

* Designed primers for pOSIP 3G amplification
* Continued plate reader experiments with heat inducible circuits
* Updated safety form

**Week 12: 180903 -180909**

* Continued plate reader experiments
* Focused on testing variations of Tlp- heat sensitive promoter
* Collaboration data was received from UVA and University of Pittsburgh
* Design logo

**Week 13: 180910 -180916**

* CTested circuit with two different promoters, tlp mflon and tlp mscarlet
* Hurricane Florence hit, school was evacuated and closed from Tuesday to Sunday
* No wetlab was able to commence
* Analyzed data from integrated circuits
* Presentation at Windsor Mead Nursing Home

**Week 14: 180917 - 180923**

* Picked up after Hurricane Florence
* Made competent flped cells
* Tested circuits transformed in pre-flped and flped cells
* Tested new ptd tag
* Experiments with integrated cells
* Created parts pages for UVA parts
* Presentation at Williamsburg Landing Nursing Home
* Poster Presentation at the Women’s Weekend

**Week 15: 180924 - 180930**

* Plate reader experiment with older parts- tsc1 mscarlet with tlp mflon
* Tested more ptd tag parts
* Continue experiments with integrated cells
* Analyzed different strength 3k3 parts
* Outbreak presentation with guest entrepreneurship speaker

**Week 16: 181001 - 181007**

* Plate reader experiment of combined circuits with tlp mflon and tlp mscarlet
* Cloned multiple circuits to be sequenced and submitted
* Presentation to Model UN

**Week 17: 181008-181017**

* Wrapped up on outstanding wetlab tasks
* Worked on Wiki!
* Ladies in the Lab

* Top
* Contact us at igem@wm.edu

Sponsors

![NEB](https://static.igem.org/mediawiki/2018/8/8f/T--William_and_Mary--NEBLogo.jpg)
![Epoch](https://static.igem.org/mediawiki/2018/a/a0/T--William_and_Mary--EpochLogo.jpg)
![Biobasic](https://static.igem.org/mediawiki/2018/7/70/T--William_and_Mary--BiobasicLogo.jpg)

2018 William & Mary iGEM


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:William and Mary/Experiments

Toggle navigation

[![](https://static.igem.org/mediawiki/2018/e/ec/T--William_and_Mary--DecodeLogo1.jpg)](https://2018.igem.org/Team%3AWilliam_and_Mary)

* [Project](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Background](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Results](https://2018.igem.org/Team%3AWilliam_and_Mary/Results)
* [Temperature Systems](https://2018.igem.org/Team%3AWilliam_and_Mary/Heat)
* [Methods and Data](https://2018.igem.org/Team%3AWilliam_and_Mary/Experiments)

- [3G Assembly](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Method Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Collaborations](https://2018.igem.org/Team%3AWilliam_and_Mary/Collaborations)
- [Parallel Circuit Testing](https://2018.igem.org/Team%3AWilliam_and_Mary/3G_Mixed)
- [3G Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
- [Protocol](https://2018.igem.org/Team%3AWilliam_and_Mary/3GProtocols)
- [Modeling](https://2018.igem.org/Team%3AWilliam_and_Mary/Model)
- [Human Practices](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Incorporating New Communities](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Incorporating_new_communities)
  * [A Statewide Standard](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/A_Statewide_Standard)
  * [Continuing the Conversation](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Continuing_the_Conversation)
  * [IHP/Silver](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices)
- Judging
  * [Medal Requirements](https://2018.igem.org/Team%3AWilliam_and_Mary/Medal%20Requirements)
  * [Measurement](https://2018.igem.org/Team%3AWilliam_and_Mary/Measurement)
  * [Demonstrate](https://2018.igem.org/Team%3AWilliam_and_Mary/Demonstrate)
  * [Best Part Collection](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
  * [Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Parts)
  * [Improve](https://2018.igem.org/Team%3AWilliam_and_Mary/Improve)
  * [Interlab](https://2018.igem.org/Team%3AWilliam_and_Mary/InterLab)
- [Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Meet the Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Attributions](https://2018.igem.org/Team%3AWilliam_and_Mary/Attributions)
  * [Notebook](https://2018.igem.org/Team%3AWilliam_and_Mary/Notebook)
  * [Protocols](https://2018.igem.org/Team%3AWilliam_and_Mary/Protocols)
  * [Safety](https://2018.igem.org/Team%3AWilliam_and_Mary/Safety)

Page Title

# Methods & Data

![](https://static.igem.org/mediawiki/2018/2/2c/T--William_and_Mary--data.png)

**Overview**

In the interest of promoting reproducible science, and enabling others to build off of our work, we have included both the precise methods used as well as the raw data (in csv format) for every experiment mentioned on our wiki. Experiments are split up by the page on which they are mentioned. For experiments performed with flow cytometry, .fcs files for both measurements and calibration beads (Spherotek RCP-30-5A, lot number: AB01) are included in the file. In all cases at least 10,000 single cell measurements were collected for flow cytometry data. Whenever used, the term biological replicates refers to a distinct colony from a transformation of the same miniprep. Whenever plate reader fluorescence data is provided, it is given in arbitrary units of fluorescence normalized to cell density (recorded in either Absorbance 600 or Absorbance 700).

**For all experiments, unless stated otherwise, the following is true:**

* Circuits used were on psb1C3
* The *E. coli* strain used was JS006 (K-12 derivative with ΔAraCΔLacI)
* The media used was M9 minimal media with 0.4% Casamino Acids and 0.4% Glucose (see protocols)
* Antibiotics in minimal media were used in the following concentrations [Chloramphenicol: 7.5mg/mL, Kanamycin: 35mg/mL]
* Transformations were plated on selective LB plates with either 25mg/mL Chloramphenicol, 50mg/mL Kanamycin or both
* 96 well plates used with the microplate reader (Synergy H1 [Biotek]) were Black Griener Fluotrack 200 flat bottom plates with the matching lid
* Microplate reader was set to linear shaking at max speed and gradient temperatures were used to prevent condensation
* Excitation/emission used for plate reader experiments was 565/593
* The channel used for flow cytometry experiments was Fl2 (S3e cell sorter [Biorad]).

**Chemical Inducible System**

**Figure 1:** Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa\_K2333427 and BBa\_K2333434 when grown at 37C and then chemically induced. Dots represent the geometric mean of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean. [Data](https://static.igem.org/mediawiki/2018/b/b4/T--William_and_Mary--180622_Data.csv)

**Figure 2:** Frozen experiment. [Data](https://static.igem.org/mediawiki/2018/0/00/T--William_and_Mary--Frozen_Vs_Not_Frozen_Data.zip)

**Figure 3:** In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 37C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then subjected to their conditions. For the data shown, those conditions were 50ng/mL ATC and 0.1mM IPTG. [Data](https://static.igem.org/mediawiki/2018/2/2a/T--William_and_Mary--Chemically_Induced_Pulse.csv)

**Figure 4:** Cells were grown at 37C in 50ng/mL ATC to induce fluorescence. At timepoint 120, the cells were split into two groups. One group was spun down and washed in media with 50ng/mL ATC, and the other group was washed in media without ATC. The cells were then left to grow again at 37C [Data](https://static.igem.org/mediawiki/2018/2/2c/T--William_and_Mary--Inducer_Spin_Down_Test.csv)

**Figure 5:** In both cases colonies were were grown at 37 C in m9 medium with 50ng/mL ATC and 0.1mM IPTG. At timepoint 40, the cells were spun down and washed in fresh m9 medium 3 times. At timepoint 80, the cells were spun down and resuspended in 50ng/mL ATC and 0.1mM IPTG m9 medium. Timepoints were taken every 20 minutes by pipetting cells into glycerol and freezing them in liquid nitrogen. [Data](https://static.igem.org/mediawiki/2018/7/75/T--William_and_Mary--Inducer_Spin_Down_Staircase.csv)

**Heat Inducible System**

**Figure 2:** In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then subjected to their conditions. For A, this was a 37C for the entire period, whereas for B this was 37C from 160 minutes to 280 minutes and 29C elsewhere. [Data](https://static.igem.org/mediawiki/2018/6/6c/T--William_and_Mary--Heat_Figure2.zip)

**Figure 3:** In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD700s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then subjected to their conditions. For A, this was a 37C for the entire period, whereas for B this was 37C from 140 minutes to 280 minutes and 29C elsewhere. [Data](https://static.igem.org/mediawiki/2018/6/64/T--William_and_Mary--Heat_Figure3.zip)

**Figure 4:** In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 37C for 1100 minutes. [Data](https://static.igem.org/mediawiki/2018/9/92/T--William_and_Mary--Heat_Figure4.zip)

**Figure 5:** In all 3 cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 30C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 39C until OD600 plateau was reached. [Data](https://static.igem.org/mediawiki/2018/7/77/T--William_and_Mary--Heat_Figure5.zip)

**Figure 6:** Different pdt constructs (1C3) were co-transformed with BBa\_K2680061 on psb3K3. Colonies were were picked into 200µL of media into a 96 well plate and cultures were grown at 30C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 39C until OD600 plateau was reached. [Data](https://static.igem.org/mediawiki/2018/c/c4/T--William_and_Mary--Heat_Figure6.csv)

**Figure 7:** K2333428 (1C3) was co-transformed with different minipreps of K2680060 and K2680061 on psb3K3. Colonies were were picked into 3mL of media and grown overnight at 30C. Cultures were then diluted 1:100 in the morning, grown for 3 hours and then 50ng/mL ATc was added. Cells were grown 4 hours and measured via flow cytometry. At the same time, cells were 1:100 diluted into fresh media and grown for 2 hours at 39C before being measured. [Part1](https://static.igem.org/mediawiki/2018/a/a5/T--William_and_Mary--1facs.zip) and [Part2](https://static.igem.org/mediawiki/2018/7/75/T--William_and_Mary--2facs.zip).

**Figure 8:** K2333428 (1C3) was transformed into JS006 cells containing the integrated constructs. 6 colonies of each transformation were then picked into 200µL of media into a 96 well plate and cultures were grown at 30C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05, and ATc at 10ng/mL and 25ng/ml was added to 3 colonies of each construct. Cells were then grown at 39C until OD600 plateau[Data](https://static.igem.org/mediawiki/2018/1/1f/T--William_and_Mary--Figure8.csv).

**Figure 9:** 3 colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 30C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 39C until OD600 plateau was reached. [Data](https://static.igem.org/mediawiki/2018/5/58/T--William_and_Mary--Heat_Figure9.csv).

**Collaboration**

**mScarlet-I RBS characterization:** Circuits composed of J23107 B0034/B0032 mScarlet-I B0015 as well as a J23107 B0034 LacI B0015 control were cloned via 3G assembly onto psb1C3. 3 colonies of each circuit were picked into LB media, grown for 3 hours at 37C, diluted to OD600 0.1 and grown 4 hours. Then circuits were measured on UVAs plate reader (with emission/excitation 565/593 and absorbance 600). Data was then normalized with respect to cell density. [Data](https://static.igem.org/mediawiki/2018/a/a5/T--William_and_Mary--Collab_data.zip)

**Flow Cytometry Protocol**

**Frozen vs Non Frozen cell tests:** 3 colonies of cells with [BBa\_K2333428](http://parts.igem.org/Part%3ABBa_K2333428) (pTet mScarlet-I pdtA) were picked into 4mL of M9 media and incubate at 37C overnight. In the morning, cells were diluted 1:100 and grown for 3 hours at 37C. Cells were diluted to a final OD600 of ~.05 in 5mL of 37C M9 and ATc was added to a final concentration of 50ng/mL. Cells were grown at 37C and 200µL time points were taken every 20 minutes (t0= immediately following inducer being added) onto ice. 50µL was filtered into ice cold PBS and measured on the FACSed immediately, while the remaining 150µL had glycerol added to a final concentration of 15%, and were then flash frozen in liquid nitrogen. Experiment was continued for 160 minutes to ensure cells remained in midlog growth. Frozen samples were stored at -80C for 3 days and then thawed on ice, 1:10 diluted into ice cold PBS and then FACSed. [Data](https://static.igem.org/mediawiki/2018/b/b0/T--William_and_Mary--Frozen.zip). FACS files ([Day1](https://static.igem.org/mediawiki/2018/8/89/T--William_and_Mary--Frozen_day1.zip) and [Day2](https://static.igem.org/mediawiki/2018/8/8a/T--William_and_Mary--Frozen_day2.zip))

**Mixed 3G Assembly**

Different transcriptional units were assembled using the parts listed on the [mixed 3G page](https://2018.igem.org/Team%3AWilliam_and_Mary/3G_Mixed) in equimolar volumes. Circuit mixture was then transformed into NEB 5-alpha cells and allowed to grow overnight at 30C. In the morning, 1 colony was picked into each well of a 96 well plate in 200µL of media. The plate was incubated at 30C until midlog growth was reached and colonies were then diluted 1:4 and grown at 37C for the remainder of the experiment. The full plate reader data can be found [here](https://static.igem.org/mediawiki/2018/c/c7/T--William_and_Mary--Mixed.zip)

**Sequencing**

All Sanger sequencing was performed by Epoch Life Sciences. All constructs submitted to the registry as well as many non submitted constructs were sequenced. A key for William and Mary circuit names as well as .ab1s can be found in the two part file [here](https://static.igem.org/mediawiki/2018/5/51/T--William_and_Mary--Sequencing1.zip) and [here](https://static.igem.org/mediawiki/2018/8/89/T--William_and_Mary--Sequencing2.zip).

* Top
* Contact us at igem@wm.edu

Sponsors

![NEB](https://static.igem.org/mediawiki/2018/8/8f/T--William_and_Mary--NEBLogo.jpg)
![Epoch](https://static.igem.org/mediawiki/2018/a/a0/T--William_and_Mary--EpochLogo.jpg)
![Biobasic](https://static.igem.org/mediawiki/2018/7/70/T--William_and_Mary--BiobasicLogo.jpg)

2018 William & Mary iGEM

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:William and Mary/Results

Toggle navigation

[![](https://static.igem.org/mediawiki/2018/e/ec/T--William_and_Mary--DecodeLogo1.jpg)](https://2018.igem.org/Team%3AWilliam_and_Mary)

* [Project](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Background](https://2018.igem.org/Team%3AWilliam_and_Mary/Description)
  + [Results](https://2018.igem.org/Team%3AWilliam_and_Mary/Results)
* [Temperature Systems](https://2018.igem.org/Team%3AWilliam_and_Mary/Heat)
* [Methods and Data](https://2018.igem.org/Team%3AWilliam_and_Mary/Experiments)

- [3G Assembly](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Method Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/3G)
  * [Collaborations](https://2018.igem.org/Team%3AWilliam_and_Mary/Collaborations)
- [Parallel Circuit Testing](https://2018.igem.org/Team%3AWilliam_and_Mary/3G_Mixed)
- [3G Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
- [Protocol](https://2018.igem.org/Team%3AWilliam_and_Mary/3GProtocols)
- [Modeling](https://2018.igem.org/Team%3AWilliam_and_Mary/Model)
- [Human Practices](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Overview](https://2018.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  * [Incorporating New Communities](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Incorporating_new_communities)
  * [A Statewide Standard](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/A_Statewide_Standard)
  * [Continuing the Conversation](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices/Continuing_the_Conversation)
  * [IHP/Silver](https://2018.igem.org/Team%3AWilliam_and_Mary/Human_Practices)
- Judging
  * [Medal Requirements](https://2018.igem.org/Team%3AWilliam_and_Mary/Medal%20Requirements)
  * [Measurement](https://2018.igem.org/Team%3AWilliam_and_Mary/Measurement)
  * [Demonstrate](https://2018.igem.org/Team%3AWilliam_and_Mary/Demonstrate)
  * [Best Part Collection](https://2018.igem.org/Team%3AWilliam_and_Mary/Part_Collection)
  * [Parts](https://2018.igem.org/Team%3AWilliam_and_Mary/Parts)
  * [Improve](https://2018.igem.org/Team%3AWilliam_and_Mary/Improve)
  * [Interlab](https://2018.igem.org/Team%3AWilliam_and_Mary/InterLab)
- [Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Meet the Team](https://2018.igem.org/Team%3AWilliam_and_Mary/Team)
  * [Attributions](https://2018.igem.org/Team%3AWilliam_and_Mary/Attributions)
  * [Notebook](https://2018.igem.org/Team%3AWilliam_and_Mary/Notebook)
  * [Protocols](https://2018.igem.org/Team%3AWilliam_and_Mary/Protocols)
  * [Safety](https://2018.igem.org/Team%3AWilliam_and_Mary/Safety)

Page Title

# Results

![](https://static.igem.org/mediawiki/2018/9/91/T--William_and_Mary--res.png)

**Construction of an Abstract IFFL Model:**

The first step in designing our decoding circuit was to examine what circuit architectures were capable of distinguishing between different types of dynamical inputs. After reviewing the literature, we found that many natural systems that utilize dynamic information encoding share a common circuit motif, that of the incoherent feedforward loop (IFFL). The IFFL is an architecture in which an input activates the production of both a reporter/output as well the production of an inhibitor of that reporter (Figure 1) [1]. Furthermore, this IFFL architecture has been shown to be capable of serving as a temporal distinguisher, meaning it can differentiate between different types of temporal (dynamic) signals.

![](https://static.igem.org/mediawiki/2018/9/95/T--William_and_Mary--iffl_architecture.png)

Figure 1: Schematic of an Incoherent Feed Forward Loop architecture. An activator (green) activates the production of a reporter/output (purple) as well as an inhibitor of the reporter (blue).

Based on our understanding that incoherent feedforward loops (IFFLs) could serve as temporal distinguishers, the first goal of our project was to replicate the model from Zhang, et al. (2016) [1]. We constructed an abstract kinetics based IFFL model, and found that IFFLs are indeed temporal classifiers. We found that when an IFFL was given an oscillatory input signal it will give qualitatively different outputs depending on the length of input signals (Figure 2). In the presence of long input signals, an IFFL will give output pulses of a fixed height, whereas in the presence of shorter input signals it will produce a staircase like output where the output concentration increases with each new input pulse.

![](https://static.igem.org/mediawiki/2018/3/38/T--William_and_Mary--pulse.png)

Figure 2: The results from our abstract IFFL model. When inputs are long (left), reporter concentration pulses at a fixed height in response to each input. When input lengths are shorter than the threshold time, each input pulse will generate a stepwise increase whose size is determined by pulse time.

The ultimate source of this behavior is the dynamics of the inhibitor; when inputs are short, the amount of inhibitor never crosses the threshold at which it efficiently degrades the output, leading to stepwise increases in the output. Conversely, when inputs are long, the inhibitor crosses the threshold and reduces the concentration of the output. Importantly for our project, these results showed that IFFLs are capable of distinguishing the temporal properties of inputs, giving different outputs depending on the length of the input signal. That means that an IFFL can provide the basis for a system that is attempting to interpret dynamic, i.e. time encoded information.

**Creating a Protease Based IFFL:**

With the knowledge that an IFFL would provide a suitable architecture for the construction of our decoder, we decided to use a previously characterized IFFL system that utilizes the mf-Lon protease system [2,3]. In this system the mf-Lon protease acts as an inhibitor by degrading a reporter with a protein degradation tag (pdt) (Figure 3).

![](https://static.igem.org/mediawiki/2017/c/c6/T--William_and_Mary--Lon_IFFL_structure.png)

Figure 3: Schematic of the dual inducible mScarlet-I-pdt mf-Lon IFFL system. Inducers act as independently tunable proxies for inputs and mf-Lon acts as an inhibitor of the reporter mScarlet-I pdt.

This is an extremely powerful and tunable system because the degradation rate constant can be tuned by using different strength pdts. By simultaneously activating the production of mf-Lon and mScarlet-I-pdt with two separate small molecule inducers, this system operates as an IFFL, with the induction of the circuit leading to the production of both the output (mScarlet-I-pdt) and its inhibitor (mf-Lon). However, by using two separate inducible systems, the production rate of the inhibitor and the output can be tuned independently of one another. Last year, work in iGEM confirmed that this system is capable of generating a pulsatile output from a continuous signal. Our first experiment therefore was to test if we could replicate this behavior on the plate reader, which would serve the dual purpose of confirming one half of the theoretical basis of our model as well as allow us to comfortably use the plate reader for future experiments involving multiple conditions. Using the registry parts K2333434 (plLac mf-Lon) and K2333428 (ptet mScarlet-I pdt#3) we were able to successfully replicate this pulsatile behavior given a constant input (Figure 4).

![](https://static.igem.org/mediawiki/2018/1/10/T--William_and_Mary--Chemically_Induced_Plate_Reader_Pulse.png)

Figure 4: Normalized to max fluorescence of the chemically induced circuits BBa\_K2333427 and BBa\_K2333434 when grown at 37C and then chemically induced with 50ng/mL ATC and 0.1mM IPTG. Dots represent the geometric mean of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean.

**Testing Short Inputs:**

We next wanted to confirm that this system would be capable of discerning between temporal inputs of different lengths. However, in order to do this we would first need to be able to determine how we could dynamically change the concentration of inducer present in solution. While our first choice would have been to use a constantly flowing microfluidics chamber along with a fluorescence microscope, we were unable to do so because of the cost involved and lack of institutional experience with such devices. Instead, we developed our own protocol in which centrifugation was used to pellet cells, allowing us to wash them and replace the media with fresh noninducer-containing media. However, we had concerns with whether this protocol would be able to remove inducer without otherwise impacting the results of experiment. One obvious concern was that since spinning down cells takes a significant amount of time, the precision of our experiments could be compromised, given that they involve the precise timing of activation periods. Other potential issues included the impact that variable temperature and the forces of centrifugation could have on gene expression.

Because of these potential pitfalls, we recognized that our first step had to be confirming that this method is valid. In our first few trials, we had issues choosing the proper speed for the centrifuge and found that our cell density was too low to form substantial cell pellets. However, with time, we were able to improve upon our procedure, eventually coming to a procedure wherein we centrifuged 50 mL of cells at 2762 RCF for 3 minutes, washed with prewarmed media, and then repeated and resuspended cells. When we tested if this method was effective at controlling the expression of a pTet inducible mScarlet by removing ATc, we found that it was effective. (Figure 5).

![](https://static.igem.org/mediawiki/2018/5/51/T--William_and_Mary--Inducer_Spin_Down_Test.png)

Figure 5: Fluorescence/OD600 (AU) measurements of the chemically induced circuits BBa\_K2333427 and BBa\_K2333434 when grown at 37C and then chemically induced with 100ng/mL ATC and 0.1mM IPTG from times 0-120. At timepoint 120, each replicate was split into two tubes and spun down for three minutes at 2762 RCF. Half of the cells were washed with media without ATC, and the other half were washed with media containing ATC. Dots represent the geometric mean of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean.

With a way to dynamically alter the input of our circuits, we next wanted to confirm the prediction that short inputs would lead to a stepwise increase in output concentration. We tested this prediction in the small molecule induced IFFL by taking single cell fluorescence measurements of an input paradigm of 40 minutes on, 40 minutes off, 40 minutes on. Using this paradigm we were able to validate the prediction of the model, showing that IFFLs do indeed function as temporal distinguishers (Figure 6).

![](https://static.igem.org/mediawiki/2018/a/a1/T--William_and_Mary--Staircase_Fixed.png)

Figure 6: Normalized (% max) fluorescence measurements of the chemically induced circuits BBa\_K2333429 (pTet mScarlet-I pdt) and BBa\_K2333434 (plLac mf-Lon) when grown at 37C and then chemically induced with 100ng/mL ATC and 0.1mM IPTG from times 0-40 and 80-120 (represented by shaded region). Dots represent the geometric mean of at least calibrated 10,000 single cell measurements of 3 distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean.

However, when we tried to extend our results we had trouble testing more cycles of inputs and shorter input pulse durations. Often times after the second centrifugation step we would obtain a small or nonexistent size pellet of cells, resulting in us have to abort the experiment. While we considered that it might be possible to scale up the size of our culture volumes, we reasoned that for this reason as well as others, the use of the chemically inducible system would limit our ability to characterize interesting dynamic patterns of input.

We evaluated a number of potential types of induction before settling on using the temperature sensitive CI (ts-CI) system. Temperature based induction offers a variety of unique benefits. First, temperature is easy to control in using standard lab equipment such as incubators and plate readers, so we would not require any equipment we don’t already have. Secondly, temperature is relevant in many in vivo systems such as those that regulate the pathogenicity of bacteria [4], meaning that our synthetic system would be potentially useful for future groups working in that area.

**Modeling a Temperature Sensitive Decoding IFFL:**

Having decided upon our new system, we next wanted to create a more biologically based mathematical model to evaluate the decoding properties of this system in particular. To do so, we developed an ODE model of our ts-CI system. We determined that our system functions as an IFFL and then sought to investigate how well our decoding circuit was able to classify different types of dynamic signals.

We first investigated how well our decoding circuit was able to distinguish between different length oscillatory inputs. Comparing the outputs of both our decoding circuit and a naive tracker (i.e. a reporter which is activated the input), we were able to determine that the decoder was superior at discerning the differences between different temporal inputs (Figure 7). Moreover, we found that the decoding circuit was particularly good at discerning even small differences in temporal inputs.

![](https://static.igem.org/mediawiki/2018/3/39/T--William_and_Mary--co1.png)

Figure 7: **Left.** Heat map comparing the euclidian distance of outputs for a given change in input pulse duration. Redder values indicate that the decoder is better able to discern the difference between the inputs. **Right.** Graphs showing the behavior of the input (top), IFFL (2nd from the top), naive model (3rd from the top), and the difference between each model for a specific change in input pulse durations (bottom). Larger differences indicate higher temporal discrimination.

Given that the decoding circuit is so sensitive to small changes in input, it would be reasonable to assume that it is also more vulnerable to noisy inputs. To test how susceptible the decoder was to noisy inputs, we added gaussian noise to the model inputs and tested the resulting output variance for a variety of different parameter regions. Surprisingly, we found that in fact the decoder has a lower output variance than the naive model when given noisy inputs (Figure 8). This is due to the proportionality effect of mf-Lon, which causes stochastic variations in the input to be weakened, leading to a less variable output. In total, our modeling shows that the decoding circuit is capable of discerning even very small temporal inputs, while still performing exponentially better in response to input variability when compared to a naive system. This implies that our system and other IFFL decoding systems represent a solution to the problem of interpreting dynamic and noisy data. To see more about how we explored the ways in which an IFFL can be made to perform superiorly to a naive circuit see our [modeling page](https://2018.igem.org/Team%3AWilliam_and_Mary/Model).

![](https://static.igem.org/mediawiki/2018/8/8e/T--William_and_Mary--MeshVariance.png)

Figure 8: Input variance as direct translation to noise. Variance of IFFL with respect to all combination of off and on time is exponentially lower than that of Naive.

**Implementation of a Temperature Sensitive IFFL:**

Having confirmed that a heat sensitive decoder would perform better than a naive tracker, and validated that the original model’s predictions, we next attempted to put our heat sensitive IFFL into practice. We designed and constructed a number of heat sensitive IFFL systems using a ts-CI repressible system with [3G Assembly](https://2018.igem.org/Team%3AWilliam_and_Mary/3G). During this testing we were able to validate that our parts were functional, yet we were unable to find a combination of parts with appropriate parameters to create a decoding IFFL. In each case, the IFFL circuits we created lead to either no degradation of reporter (a too high reporter/mf-Lon ratio), no fluorescence (a too high mf-Lon/reporter ratio) or cell death (too high mf-Lon).

We attempted to solve this problem in a number of different ways. We tried combining our circuits onto a single plasmid construct, genomically integrating constructs, and adding a strong degradation tag to mf-Lon. However, none of those approaches were successful in obtaining a temperature sensitive IFFL with the appropriate parameters. We next decided to shift to an alternative temperature sensitive system utilizing the temperature sensitive repressor TlpA and its cognate promoter operators. However, despite many iterative design build test cycles, we were unable to get any of these approaches to produce IFFLs which outputted the characteristic pulse when activated. See [our page on temperature inducible systems](https://2018.igem.org/Team%3AWilliam_and_Mary/Heat).

Moving forward, we believe that chemically induced systems show the most promise. Our preliminary data indicates that with the proper equipment (microfluidics), chemically inducible IFFLs may serve as a tool to distinguish temporal inputs. In addition to chemical induction, however, there is potential for other types of inducible IFFLs to act as decoders, including optogenetic systems and other temperature sensitive systems such as those utilizing riboswitches or RNA thermometers.

**References**

[1] Zhang C, Tsoi R, Wu F, You L (2016) Processing Oscillatory Signals by Incoherent Feedforward Loops. PLoS Comput Biol 12(9): e1005101. doi:10.1371/journal.pcbi.1005101

[2] D Ewen Cameron and James J Collins. Tunable protein degradation in bacteria. Nature biotechnology, 32(12):1276–1281, 20

[3] https://2017.igem.org/Team:William\_and\_Mary/Dynamic\_Control

[4] Gal-Mor O, Valdez Y, Finlay BB. The temperature-sensing protein TlpA is repressed by PhoP and dispensable for virulence of Salmonella enterica serovar Typhimurium in mice. Microbes Infect / Institut Pasteur. 2006;8:2154–62. doi:10.1016/j.micinf.2006.04.015.

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