iGEM 2018 — Team Northwestern

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# Team:Northwestern/Notebook

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[HOME](https://2018.igem.org/Team%3APurdue)

TEAM

[Team Members](https://2018.igem.org/Team%3APurdue/Team)
[Collaborations](https://2018.igem.org/Team%3APurdue/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3APurdue/Description)
[Design](https://2018.igem.org/Team%3APurdue/Design)
[Experiments](https://2018.igem.org/Team%3APurdue/Experiments)
[Notebook](https://2018.igem.org/Team%3APurdue/Notebook)
[InterLab](https://2018.igem.org/Team%3APurdue/InterLab)
[Model](https://2018.igem.org/Team%3APurdue/Model)
[Results](https://2018.igem.org/Team%3APurdue/Results)
[Demonstrate](https://2018.igem.org/Team%3APurdue/Demonstrate)
[Improve](https://2018.igem.org/Team%3APurdue/Improve)
[Attributions](https://2018.igem.org/Team%3APurdue/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3APurdue/Parts)
[Basic Parts](https://2018.igem.org/Team%3APurdue/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3APurdue/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3APurdue/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3APurdue/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3APurdue/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3APurdue/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3APurdue/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3APurdue/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3APurdue/Hardware)
[Measurement](https://2018.igem.org/Team%3APurdue/Measurement)
[Model](https://2018.igem.org/Team%3APurdue/Model)
[Plant](https://2018.igem.org/Team%3APurdue/Plant)
[Software](https://2018.igem.org/Team%3APurdue/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Purdue)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:Northwestern/Experiments

Northwestern Template

Northwestern Template

[Northwestern](https://2018.igem.org/Team%3ANorthwestern)

Human Practices

[Overview](https://2018.igem.org/Team%3ANorthwestern/Human_Practices)
[Outreach](https://2018.igem.org/Team%3ANorthwestern/Outreach)
[Interviews](https://2018.igem.org/Team%3ANorthwestern/Interviews)
[Collaborations](https://2018.igem.org/Team%3ANorthwestern/Collaborations)

Parts

[Overview](https://2018.igem.org/Team%3ANorthwestern/Parts)

Lab

[Experiments](https://2018.igem.org/Team%3ANorthwestern/Experiments)
[Modeling](https://2018.igem.org/Team%3ANorthwestern/Model)
[Results](https://2018.igem.org/Team%3ANorthwestern/Results)
[Notebook](https://2018.igem.org/Team%3ANorthwestern/Lab)
 [Safety](https://2018.igem.org/Team%3ANorthwestern/Safety)
 [Interlab](https://2018.igem.org/Team%3ANorthwestern/InterLab)

Project

[Description](https://2018.igem.org/Team%3ANorthwestern/Description)
[Design](https://2018.igem.org/Team%3ANorthwestern/Design)
[Medal Criteria](https://2018.igem.org/Team%3ANorthwestern/Medals)

Team

[About Us](https://2018.igem.org/Team%3ANorthwestern/Team)
[Attributions](https://2018.igem.org/Team%3ANorthwestern/Attributions)

[Home](https://2018.igem.org/Team%3ANorthwestern)

Home

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![](https://static.igem.org/mediawiki/2018/9/9c/T--Northwestern--Experiments.png)

### What We Aimed For

With only theoretical knowledge from literature, our first ambitious goal was to be able to produce the reporter protein sfGFP (or a colorimetric enzyme) completely in vitro and at least be able to tell analyte concentration semi-quantitatively. Our other goals included high sensitivity, easy for citizens to use, precise and consistent results, generating results in less than 4 hours
and holding no risk of introducing/handling/shipping living cells in environment.

### Our reporter construct

We had two separate platforms on which we constructed our biosensors. The first one is the Two-Plasmid Repressor System and the other is a Two-Plasmid Activator System.
The repressor plasmid constitutively produces repressor protein which is regulated by the analyte. The reporting plasmid for this system, on the other hand, contains an operator site which is regulated by the repressor protein. Therefore, it only has an output in the absence of the repressor protein.

Our metal sensors are both two plasmid repressor systems. Since neither of our construct systems have been well-characterized in literature, we decided that starting out with a more well-characterized system would be beneficial. This is why we decided to start out by testing and engineering a system to sense AHL. AHL is a signal molecule used in quorum sensing as a regulatory mechanism by which gram-negative bacteria control gene expression in response to population density. The importance and well-characterization of AHL sensing made it a prime target for us to analyze.

![](https://static.igem.org/mediawiki/2018/6/63/T--Northwestern--Cr.jpg)

Figure 1: A Chromium-Detecting Two-Plasmid System

![](https://static.igem.org/mediawiki/2018/5/59/T--Northwestern--Pb.jpg)

Figure 2: A Lead-Detecting Two-Plasmid System

We started out by basing our constructs on a system characterized by the Jewett Lab at Northwestern. These were viable for a cell-free system, but they were not well optimized for such a platform. Our first aim was to optimize this system for better cell-free expression.

On the first plasmid, we replaced the endogenous pTet with a T7 promoter, because the latter is stronger, unregulated, processive polymerase, which is important to us because we want LuxR to be present in excess for accurate sensing. For the same reason we couldn’t use a processive polymerase on the second plasmid, because we didn’t want our system to leak in the off state. Additionally, we engineered in a PHP stability hairpin to our second plasmid, which helps prevent exonuclease activity, which should significantly increase the activity of our biosensor.

![](https://static.igem.org/mediawiki/2018/e/e1/T--Northwestern--Luxr1.jpg)

Figure 3: Unoptimized AHL Detection System

![](https://static.igem.org/mediawiki/2018/5/5e/T--Northwestern--Luxr2.jpg)

Figure 4: Optimized AHL Detection System

### Experimental setup

Our first step was to test our unoptimized systems in conjunction with optimized systems and compare our results and verify the functionality of our systems. Subsequently, we tested different analyte concentrations. For further information, see the [Results Page](https://2018.igem.org/Team%3ANorthwestern/Results).

### References

**1.** Adam D Silverman, Nancy Kelley-Loughnane, Julius B Lucks, Michael C Jewett
bioRxiv 411785; doi: https://doi.org/10.1101/411785
**2.** Pellinen, Teijo, et al. “A Cell-Free Biosensor for the Detection of Transcriptional Inducers Using Firefly Luciferase as a Reporter.” Analytical Biochemistry, vol. 330, no. 1, 13 May 2004, pp. 52–57., doi:10.1016/j.ab.2004.03.064.

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# Team:Northwestern/Results

Northwestern Template

Northwestern Template

[Northwestern](https://2018.igem.org/Team%3ANorthwestern)

Human Practices

[Overview](https://2018.igem.org/Team%3ANorthwestern/Human_Practices)
[Outreach](https://2018.igem.org/Team%3ANorthwestern/Outreach)
[Interviews](https://2018.igem.org/Team%3ANorthwestern/Interviews)
[Collaborations](https://2018.igem.org/Team%3ANorthwestern/Collaborations)

Parts

[Overview](https://2018.igem.org/Team%3ANorthwestern/Parts)

Lab

[Experiments](https://2018.igem.org/Team%3ANorthwestern/Experiments)
[Modeling](https://2018.igem.org/Team%3ANorthwestern/Model)
[Results](https://2018.igem.org/Team%3ANorthwestern/Results)
[Notebook](https://2018.igem.org/Team%3ANorthwestern/Lab)
 [Safety](https://2018.igem.org/Team%3ANorthwestern/Safety)
 [Interlab](https://2018.igem.org/Team%3ANorthwestern/InterLab)

Project

[Description](https://2018.igem.org/Team%3ANorthwestern/Description)
[Design](https://2018.igem.org/Team%3ANorthwestern/Design)
[Medal Criteria](https://2018.igem.org/Team%3ANorthwestern/Medals)

Team

[About Us](https://2018.igem.org/Team%3ANorthwestern/Team)
[Attributions](https://2018.igem.org/Team%3ANorthwestern/Attributions)

[Home](https://2018.igem.org/Team%3ANorthwestern)

# Results Unoptimized Plasmid Assay We first tested our unoptimized sensor with sfGFP as reporter gene in vitro to confirm that the sensor is functional. A concentration of 100 nM of AHL was used. The results are shown in figure 1. We observed a reaction approximately 300 minutes after addition of AHL. The reaction could clearly be distinguished from the negative control and the fluorescence signal increased up to 90 a.u. Nevertheless, our fold induction was around 5, which could be further increased with optimization. This fluorescence is also visually indistinguishable from the negative control without the use of a plate reader.

![](https://static.igem.org/mediawiki/parts/e/e1/T--Northwestern--graph1.png)

Figure 1. - Unoptimized Plasmid Assay

### Optimized Plasmid Assays

Next, we performed the same experiment using our optimized plasmid. We used a concentration of 100 nM AHL, just like our first experiment. We also used a positive control for our experiment which was a sfGFP with an upstream T7. Our negative control was a blank extract. The results can be found in figure 2A. Our optimized system outperformed the unoptimized system in two ways: the absolute fluorescence of the ON state increased (Figure 2A), as did the fold-induction (the ratio of ON to OFF states) (Figure 2B).

![](https://static.igem.org/mediawiki/parts/c/c3/T--Northwestern--graph.png
) ![](https://static.igem.org/mediawiki/parts/9/9a/T--Northwestern--Fold.png
)

Figure 2A & 2B. - Optimized Plasmid Assay & Fold Induction Graph

### AHL Titration

Subsequently, we tested different AHL concentrations. The kinetics of how our sensors response to different AHL concentrations is shown in figure 3A. Since we had received such a low signal in our previous experiments for an AHL concentration of 100 nM we decided to use higher concentrations for this experiment. The first two hours show a strong increase in fluorescence. After that, the increase in fluorescence became slower. We could also show that the adding different concentrations of AHL has effects on the transcription levels of sfGFP. Compared to the former fluorescence levels our optimized device showed higher levels of fluorescence. This range of AHL that we used was out of the range of linear fluorescence increase predicted by our model. For information about our model visit our [Modeling page](https://2018.igem.org/Team%3ANorthwestern/Model). The extract that we used for this experiment also seems to be higher, with better fold induction and fluorescence that is visible to the naked eye. If we had more time we would conduct this experiment with lower concentrations of AHL to obtain data for a wider range of concentrations. After a concentration of 20 μM of AHL, the fluorescence decreased drastically. Since AHL was dissolved in Ethanol, we can assume that after this concentration, the AHL became toxic to our system. If we had more time, we would apply what we learned to our Chromium and Lead systems. A more detailed analysis of our future directions is also listed below.

![](https://static.igem.org/mediawiki/parts/3/3e/T--Northwestern--graph2.png
) ![](https://static.igem.org/mediawiki/parts/2/2f/T--Northwestern--graph3.png)

Figure 3A & 3B. - AHL Titration Data & Fold Induction Graph

### Achievements

• Able to obtain results in under 4 hours.

• Successfully showed that our system responded to the presence of AHL.

• Showed that all of pLux, LuxR and AHL are needed for GFP production.

• Further optimizations can be done to reduce the leak in the off state.

• A positive correlation between AHL concentration and fluorescence was established.

• The maximum concentration of AHL detectable before our system got poisoned was 20 μΜ.

• Successfully cloned and sequenced ChrB and ChrP.

• Our experiments could not confirm a chromate dependent interaction of ChrB and Pchr.

• Successfully cloned and sequenced PbrR.

• Cloning of PbrAP was unsuccessful.

• Our experiments could not confirm a chromate dependent interaction of PbrR and PbrAP.

• We were lacking time for further in vitro characterizations and experimental setups.

### Future Directions

Although we have made great progress towards creating this biosensor, we acknowledge that there are still improvements that could made to develop it further. Initially, our team planned to improve upon the Biefeld team’s work in 2015 by extending what they were able to develop in vivo to cell-free. The team was able to create whole-cell sensors that detected numerous heavy metals, but were unable to transition to in vitro. We were able replicate the potential of our system with our modified LuxR/pLux system, a well-known circuit within the synthetic biology community. However, due to time constraints, we were unable to move the lead and chromium sensors toward cell-free. With more time, we are optimistic our circuits would work with our specially-designed, cell-free vectors.

From there, we would be able to transition from in vitro to cell-free and blot the systems on paper. Although this transition has been proven to be difficult, our collaboration with Purdue and our extensive network of Northwestern faculty that specialize in cell-free reactions drive us to believe our system would function correctly. We have altered the repressor plasmids to contain T7 promoters, a type of constitutive promoter, and added a stability hairpin to facilitate a better output before transitioning to cell-free.

One of the major improvements that we would like to make is adapting the sensor to offer more information than a simple yes or no output regarding the presence of the heavy metal. This lead our team to attend a “Biosensor Practicum” at Northwestern, where we were exposed to the world of possibilities that synthetic biology, namely biosensors offer. Specifically, the work of Dr. Mark Styczynski and his in vitro detection of zinc changed the way we saw our sensor impacting the world. His team developed a biosensor for zinc that had a tri-color output based on the concentration of the metal present. Thus, the sensor met the design requirements that citizens should be able to easily interpret results, the results should be present within a couple hours, and MetaSense should have a high sensitivity. We were fascinated with the idea of having both the immediate feedback of a colorimetric output as well as the detailed specificity of being able to determine multiple ranges of the metal’s concentration. Thus, we would develop this sensor further to include those characteristics, time-permitting, in order to offer our user’s an easy-to-use and descriptive sensor.

Once the entire sensor was developed, the next aspect we would need to improve upon is the distribution of the system. The benefit to cell-free is that storage and transportation requirements are low, meaning we could partner with a distributor to provide MetaSense to citizens through their mail. In theory, we could partner with an existing paper-based detection company such as Micro Essential Laboratory and have them ship and distribute our tests across the US. Another option would be to partner with the Evanston water treatment plant and offer our sensor to citizens whose water is treated by the plant. By making these changes and addressing these future developments, we would be able to provide an accurate, simple, and efficient sensor for detecting heavy metal presence.