iGEM 2019 — Team Virginia

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# Team:Virginia/Notebook

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TEAM

[Team Members](https://2019.igem.org/Team%3AVirginia/Team)
[Collaborations](https://2019.igem.org/Team%3AVirginia/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AVirginia/Description)
[Design](https://2019.igem.org/Team%3AVirginia/Design)
[Experiments](https://2019.igem.org/Team%3AVirginia/Experiments)
[Notebook](https://2019.igem.org/Team%3AVirginia/Notebook)
[Contribution](https://2019.igem.org/Team%3AVirginia/Contribution)
[Results](https://2019.igem.org/Team%3AVirginia/Results)
[Demonstrate](https://2019.igem.org/Team%3AVirginia/Demonstrate)
[Improve](https://2019.igem.org/Team%3AVirginia/Improve)
[Attributions](https://2019.igem.org/Team%3AVirginia/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AVirginia/Parts)
[Basic Parts](https://2019.igem.org/Team%3AVirginia/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AVirginia/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AVirginia/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AVirginia/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AVirginia/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AVirginia/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AVirginia/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AVirginia/Hardware)
[Measurement](https://2019.igem.org/Team%3AVirginia/Measurement)
[Model](https://2019.igem.org/Team%3AVirginia/Model)
[Plant](https://2019.igem.org/Team%3AVirginia/Plant)
[Software](https://2019.igem.org/Team%3AVirginia/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Virginia)

# 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:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [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:Virginia/Experiments

TRANSFOAM

[![](https://static.igem.org/mediawiki/2019/a/a1/T--Virginia--2019_Polished_Logo.png)](https://2019.igem.org/Team%3AVirginia)

![](https://static.igem.org/mediawiki/2019/8/8c/T--Virginia--2019_team.png)TEAM

[Team](https://2019.igem.org/Team%3AVirginia/Team)
[Attributions](https://2019.igem.org/Team%3AVirginia/Attributions)

![](https://static.igem.org/mediawiki/2019/9/9e/T--Virginia--2019_project.png)PROJECT

[Description](https://2019.igem.org/Team%3AVirginia/Description)
[Design](https://2019.igem.org/Team%3AVirginia/Design)
[Experiments](https://2019.igem.org/Team%3AVirginia/Experiments)
[Model](https://2019.igem.org/Team%3AVirginia/Model)
[Safety](https://2019.igem.org/Team%3AVirginia/Safety)

![](https://static.igem.org/mediawiki/2019/3/37/T--Virginia--2019_results.png)RESULTS

[Results](https://2019.igem.org/Team%3AVirginia/Results)
[Future Directions](https://2019.igem.org/Team%3AVirginia/Improve)
[Demonstrate](https://2019.igem.org/Team%3AVirginia/Demonstrate)

![](https://static.igem.org/mediawiki/2019/5/54/T--Virginia--2019_parts.png)PARTS

[Parts Collection](https://2019.igem.org/Team%3AVirginia/Parts)

![](https://static.igem.org/mediawiki/2019/d/de/T--Virginia--2019_outreach.png)OUTREACH

[Integrated Human Practices](https://2019.igem.org/Team%3AVirginia/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AVirginia/Public_Engagement)
[Collaborations](https://2019.igem.org/Team%3AVirginia/Collaborations)
[Mid-Atlantic Meetup](https://2019.igem.org/Team%3AVirginia/mid-atlantic)

![](https://static.igem.org/mediawiki/2019/f/f2/T--Virginia--2019_awards.png)AWARDS

[Awards](https://2019.igem.org/Team%3AVirginia/awards)
[Medals](https://2019.igem.org/Team%3AVirginia/medals)

![](https://static.igem.org/mediawiki/2019/0/0b/T--Virginia--2019_experiments.png)

* [1
  DNA Synthesis](#dna)
* [2
  Assembly & Plasmid
             Confirmation](#assembly)
* [3
  PHB Production
             & Extraction](#phb)

To see the protocols we used during experimentation, click [here](https://static.igem.org/mediawiki/2019/2/2a/T--Virginia--protocols.pdf).
To see the team’s lab log, click [here](https://static.igem.org/mediawiki/2019/a/a4/T--Virginia--LabNotebook.pdf).

## DNA Synthesis

**Design using Teselagen**

Using Teselagen, a DNA design and plasmid assembly software, our team designed out BioBricks for Golden Gate and Gibson assembly respectively.

![](https://static.igem.org/mediawiki/2019/8/8e/T--Virginia--2019_dnasynthesis1.png)![](https://static.igem.org/mediawiki/2019/0/09/T--Virginia--2019_dnasynthesis2.png)

***pha* Plasmid**

Our plasmid that encodes genes for the biosynthesis of polyhydroxybutyrate was designed with 3 essential genes, *phaA*, *phaB*, and *phaC*. A customized synthetic RBS site was inserted before each coding region, to optimize translation. The genes *phaA* ([BBa\_K3192015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3192015)), *phaB* ([BBa\_K3192016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3192015)), and *phaC*([BBa\_K3192017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3192015)) encode for enzymes essential for the conversion of acetyl coa into polyhydroxybutyrate (PHB). *phaA* encodes for b-Ketoacyl-CoA thiolase. *phaB* encodes for b-Ketoacyl-CoA reductase. *phaC* encodes for poly(3-hydroxybutyrate) polymerase. This plasmid was maintained with chloramphenicol resistance.

***paa* Pathway**

We chose a chassis organism, *E. coli* TG1, that constitutively expresses the paa pathway. Implementing the *paa* pathway through a plasmid into a chassis organism would be too large because there are 11 genes essential to the conversion of phenylacetic acid to acetyl-coA. We chose TG1 so that we did not have to incorporate this pathway into our chassis.

***sty* plasmid**

Our second plasmid encodes for the biodegradation of styrene into phenylacetic acid. The genes *styA*, *styB*, *styC*, *styD*, and *styE* are essential for proteins and enzymes that uptake styrene into a cell and degrade it to phenylacetate. *styA* ([BBa\_K3192015](http://parts.igem.org/Part%3ABBa_K3192015)) Codes for the styrene monooxygenase subunit A, which catalyzes the conversion of styrene to styrene oxide. *styB* ([BBa\_K3192016](http://parts.igem.org/Part%3ABBa_K3192016)) codes for styrene monooxygenase subunit B, which catalyzes the conversion of styrene to styrene oxide. *styC* ([BBa\_K3192017](http://parts.igem.org/Part%3ABBa_K3192017)) codes for epoxystyrene isomerase which catalyzes the reaction from styrene oxide to phenylacetaldehyde. *styD* ([BBa\_K3192018](http://parts.igem.org/Part%3ABBa_K3192018)) codes for phenylacetaldehyde dehydrogenase which catalyzes the reaction from phenylacetaldehyde to phenylacetic acid. *styE* ([BBa\_K3192019](http://parts.igem.org/Part%3ABBa_K3192019)) codes for the putative styrene transporter which is a membrane bound protein that aids in the transport of styrene across the cellular membrane and into the cell. This plasmid was maintained with ampicillin resistance. Using two different antibiotic resistance genes, one for each plasmid, allowed for both to be maintained within a single cell.

![](https://static.igem.org/mediawiki/2019/4/40/T--Virginia--2019_pathway.png)

## Assymbly & Plasmid Confirmation

**Golden Gate**

Golden gate assembly was used to assemble both the *pha* and the *sty* plasmids. All of our synthesized DNA constructs were designed to come together in the exact order that was desired, through the addition of a 5 base pair recognition sequence. This sequence is complementary on each end with the adjacent overlapping sequence.

![](https://static.igem.org/mediawiki/2019/b/bd/T--Virginia--2019_goldengate.png)

Retrieved from [1](https://www.neb.com/products/e1600-neb-golden-gate-assembly-mix#Product%20Information).

The PHA plasmid was cloned with fragments that were synthesized by Twist and IDT to have the desired restriction sites on either end of the parts. These parts were then assembled into the vector pGGA provided by NEB. This has chloramphenicol resistance and was grown on LB - Agar plates. The *sty* plasmid was also synthesized by Twist and IDT to have the correct restriction sites on either side of the fragments. This was assembled with the vector (pGGEV\_3\_Linker) that contained the ampicillin resistance gene. Both plasmids have different origins of replication to ensure that they can both be maintained during replication.

**Gibson Assembly**

Gibson Assembly was attempted for both parts. Due to the amount of parts and the nature of Gibson assembly, it was difficult to assemble the plasmid in the correct order and prevent reannealing in one reaction tube. This is the trademark for Gibson Assembly, thus we found that it was not a viable solution for ensuring correct plasmid formation. Many attempts of various methods were tried to prevent reannealing during the Gibson Assembly reaction, but for the amount of parts in the tube, it was difficult to ensure all the parts formed in the correct order, and all the pieces were contained within the plasmid.

![](https://static.igem.org/mediawiki/2019/d/d8/T--Virginia--2019_gibson.png)

Pictures taken from GenScript GenBuilder Plus Cloning Kit, that our team used to attempt Gibson assembly.

**Restriction Enzyme Digest and Gel Electrophoresis**

To confirm the identity of our assembled plasmids, the plasmid was digested with 1 to 2 enzymes that would produce unique bands when run on a gel. The digested plasmid was run on a gel to confirm its identity to see if the bands matched up with what was expected from the digest. Teselagen was again used to analyze the expected result of the digest and our resulting digest.

![](https://static.igem.org/mediawiki/2019/8/8e/T--Virginia--2019_gel1.png)

Teselegan Gel Electrophoresis simulation results of *pha* plasmid cut with restriction enzyme EagI

![](https://static.igem.org/mediawiki/2019/2/25/T--Virginia--2019_gel2.png)

Gel Electrophoresis results of *pha* plasmid cut with restriction enzyme Eagl

**Sequencing the Plasmids**

Once a gel was run and indicated that our plasmid likely assembled correctly, the plasmid was sent out for sequencing. Primers for each plasmid were designed to ensure that high quality results would be obtained from the overlaps in assembly to ensure that one piece assembled adjacent to its next piece. The primer for each overlap began sequencing ~150 base pairs upstream from the overlap.

**Electroporation for Chassis Assembly**

Electrocompetent TG1 cells were transformed with the assembled *pha* plasmid using electroporation. In order to insert the second *sty* plasmid into the TG1 cell containing the *pha* plasmid, the TG1 cells had to be made electrocompetent. Antibiotic selection was used to ensure the first *pha* plasmid was still retained within the chassis. Once we confirmed that the cells were again electrocompetent, and the *pha* plasmid was retained, the bacteria were transformed with the assembled *sty* plasmid using electroporation. These cells were then selected with ampicillin and chloramphenicol dual resistance agar plates.

![](https://static.igem.org/mediawiki/2019/e/e4/T--Virginia--2019_chassy1.png)

Our two plasmids in the TG1 chassis growing on a dual resistance chloramphenicol and ampicillin plate.

## PHB Production & Extraction

**Integrating Styrene**

![](https://static.igem.org/mediawiki/2019/a/ac/T--Virginia--2019_flask1.png)Styrene cannot be added directly to the medium the cells are growing in, so an alternative method to introducing styrene needed to be used. Consulting the literature, there were various methods to introduce styrene to our cells. Two promising methods were vaporization1 and partitioning.2 Partitioning was chosen due to the ability to measure the amount of styrene uptake with gas chromatography.

![](https://static.igem.org/mediawiki/2019/2/2c/T--Virginia--2019_flask2.png)Styrene was dissolved in dioctyl phthalate, an organic solvent, which was then placed above the M9 medium in a biphasic mixture.
The method of partitioning is done by the solute, styrene, distributing through two immiscible solvents, dioctyl phthalate and M9 medium. When shaken, the styrene molecules will partition into the water molecules of the M9 medium. Because styrene is very insoluble to water, only a small, but sufficient, amount will reach the cell membranes. There is an equilibrium of styrene concentration in both solvents, and when styrene molecules are taken into the cell, this change in concentration will continue to push styrene towards equilibrium as it is metabolized. This will provide the cells with a consistent influx of styrene. These styrene molecules can then be transported into the cell using the membrane-bound transporter protein translated by the styE gene. Once taken into the cells, they can begin breaking down the styrene into phenyl acetic acid.

**Red Nile Staining**

Red Nile is a qualitative means to determine PHB production within a culture of cells.3 Red Nile is effective in measuring lipid droplets by fluorescence microscopy and flow cytofluorometry. The fluorescence is conditional on the presence of strong hydrophobic regions, hence why it is ideal for detecting PHBs. Our team utilized Red Nile staining and fluorescence microscopy to preliminarily assay PHB production within our chassis to confirm the function of our PHB plasmid.

![](https://static.igem.org/mediawiki/2019/4/41/T--Virginia--2019_rednile.png)

**SDS Extraction**

Sodium Dodecyl Sulfate (SDS) is a common chemical used for cell digestion and washing. PHB producing cells were spun down and SDS extraction was performed on the pellet to separate the cell pellet from the PHB. SDS digests the cell such that PHB can be separated from It is important to note that the SDS extraction does not physically separate the cell debris and PHB, only lyses the cell.4

**Chloroform with Acetone**

We first used hot acetone (60°C) to lyse the cells open, exposing the PHB contained within the cell. Chloroform was then added to this mixture. Chloroform is an efficient solvent for PHB, and can be used to purify it from a solution. Dissolution of the pellet in chloroform followed by filtration allowed PHB to be separated from cell debris. PHB could then be reprecipitated into solution with the addition of methanol. Repetition of this procedure multiple times was found to increase purity of PHB extraction.5

**SDS and Chloroform in Conjunction**

Using a sequential extraction of SDS and chloroform was implemented by our team to attempt to lyse the cells with SDS extraction, and then purify the PHBs from solution with chloroform extraction. The conjunctive procedure allowed efficient separation of cell debris and PHB as well as increased PHB purity from extraction.

**Sonication and centrifugal differentiation**

Given the environmental focus of our team, SDS, but especially chloroform extraction were not appealing to us, as both involve potent and harmful chemicals. Our team searched for another protocol for PHB extraction with lower environmental impacts, and eventually developed our own using the density difference of PHB and organic cell debris to separate the two. Through sonication, lysing the cells using sound energy, we broke the cells apart to release PHB. Applying the pellet obtained from sonication to a sucrose solution with a density of 1.23 g/mL the PHBs could be separated from the cell pellet through density differential centrifugation. The sucrose density was chosen because the density of PHBs is 1.25 g/mL and the density of the average cell debris is 1.10 g/mL, so by centrifuging the pellet in a solution with a density slightly less dense than PHB the two solids can be effectively separated.

This protocol is outlined in more depth in our [protocols handbook](https://static.igem.org/mediawiki/2019/2/2a/T--Virginia--protocols.pdf).
This protocol has undergone refinement, but is still far from perfection. We recognize that there were likely impurities in our PHB samples of cell debris dense enough to pellet with it, and PHB that was likely light enough not to pellet with the rest of the pellet.

# References

1. Ward, P. G., Goff, M., Donner, M., Kaminsky, W. & O’Connor, K. E. A Two Step Chemo-biotechnological Conversion of Polystyrene to a Biodegradable Thermoplastic. *Environ. Sci. Technol.*  **40**, 2433–2437 (2006).
2. Osswald, P., Baveye, P. & Block, J. C. Bacterial influence on partitioning rate during the biodegradation of styrene in a biphasic aqueous-organic system. *Biodegradation* **7**, 297–302 (1996).
3. Tyo, K. E., Zhou, H. & Stephanopoulos, G. N. High-Throughput Screen for Poly-3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803. *Appl. Environ. Microbiol.*  **72**, 3412–3417 (2006).
4. Chen, Q. & Zhang, L. H. Study on Synthesis of PHB by Moderate Halophile and Aqueous Extraction of PHB. *Applied Mechanics and Materials* (2014) doi:10.4028/www.scientific.net/AMM.448-453.160.
5. Hahn, S. K., Chang, Y. K. & Lee, S. Y. Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli. *Appl. Environ. Microbiol.* **61**, 34–39 (1995).

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# Team:Virginia/Results

TRANSFOAM

[![](https://static.igem.org/mediawiki/2019/a/a1/T--Virginia--2019_Polished_Logo.png)](https://2019.igem.org/Team%3AVirginia)

![](https://static.igem.org/mediawiki/2019/8/8c/T--Virginia--2019_team.png)TEAM

[Team](https://2019.igem.org/Team%3AVirginia/Team)
[Attributions](https://2019.igem.org/Team%3AVirginia/Attributions)

![](https://static.igem.org/mediawiki/2019/9/9e/T--Virginia--2019_project.png)PROJECT

[Description](https://2019.igem.org/Team%3AVirginia/Description)
[Design](https://2019.igem.org/Team%3AVirginia/Design)
[Experiments](https://2019.igem.org/Team%3AVirginia/Experiments)
[Model](https://2019.igem.org/Team%3AVirginia/Model)
[Safety](https://2019.igem.org/Team%3AVirginia/Safety)

![](https://static.igem.org/mediawiki/2019/3/37/T--Virginia--2019_results.png)RESULTS

[Results](https://2019.igem.org/Team%3AVirginia/Results)
[Future Directions](https://2019.igem.org/Team%3AVirginia/Improve)
[Demonstrate](https://2019.igem.org/Team%3AVirginia/Demonstrate)

![](https://static.igem.org/mediawiki/2019/5/54/T--Virginia--2019_parts.png)PARTS

[Parts Collection](https://2019.igem.org/Team%3AVirginia/Parts)

![](https://static.igem.org/mediawiki/2019/d/de/T--Virginia--2019_outreach.png)OUTREACH

[Integrated Human Practices](https://2019.igem.org/Team%3AVirginia/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AVirginia/Public_Engagement)
[Collaborations](https://2019.igem.org/Team%3AVirginia/Collaborations)
[Mid-Atlantic Meetup](https://2019.igem.org/Team%3AVirginia/mid-atlantic)

![](https://static.igem.org/mediawiki/2019/f/f2/T--Virginia--2019_awards.png)AWARDS

[Awards](https://2019.igem.org/Team%3AVirginia/awards)
[Medals](https://2019.igem.org/Team%3AVirginia/medals)

![](https://static.igem.org/mediawiki/2019/4/4e/T--Virginia--2019_results1.png)

* [1
  Synthetic RBS
             Collection](#rbs)
* [2
  Sty Plasmid
            Characterization](#sty)
* [3
  PHB Production](#phb)
* [4
  Summary](#summary)

## Synthetic RBS Collection

When expressing gene clusters in a foreign chassis, the endogenous ribosomal binding sites (RBS) are often included in the sequence information provided by NCBI. Although these RBS are sufficient, they may not provide optimal translational efficiency. Synthetic RBSs provide a solution to increase translation initiation rates by using software to design a RBS with a linear secondary structure that allows for optimal ribosomal binding and translation. Our team’s project required enhanced translation of our coding sequences; therefore we chose to optimize all RBSs for high translation initiation rates. Using DeNovo DNA, our team created synthetic RBSs that ran thousands of iterations to create substantially elevated translation initiation rates. Our parts collection of nine synthetic RBSs is a toolbox for assaying translation in the context of different biological devices.

![](https://static.igem.org/mediawiki/2019/a/a1/T--Virginia--2019_gears.png)

## *sty* Plasmid Characterization

Our team encountered difficulties assembling our sty plasmid. We believe that this was primarily due to the four inserts that were cloned into a vector using Golden Gate Assembly, as assembly success decreases with increasing number of inserts. After our plasmid had been assembled, we transformed it into *E. coli* TG1 expressing our pha plasmid, and into *E. coli* DH5-ɑ. With the DH5-ɑ, we began culturing them in M9 growth media supplemented with 2mL styrene and 400µL of glucose,only 400µL of glucose, and 2mL styrene. 400µL of glucose was chosen for addition because it is not enough to solely sustain bacterial growth.2 Growth was measured through OD taken periodically. These ODs taken over 56 hours display that styrene is not a sufficient sole carbon source for growth, indicating that the sty plasmid is not functional.

![](https://static.igem.org/mediawiki/2019/b/b9/T--Virginia--graph.png)

## PHB Production

**Pha plasmid induced by IPTG**

![](https://static.igem.org/mediawiki/2019/2/2a/T--Virginia--2019_iptg.png)Our team successfully constructed a plasmid that is capable of producing PHBs given simple carbon sources, such as glucose, which we used for preliminary functional testing of our BioBrick’s (BBa\_K3192031) function. To determine qualitatively if BBa\_K3192031 worked, we performed Red Nile staining to test for PHA presence.1

Our team also wanted visual insight of PHB production in a cell on the cellular level. With the help of the University of Virginia Medical School Microscopy Center our team gather Electron Microscopy images of the PHB production. Due to time constraints our EM pictures were not obtained in time for upload to our wiki, but we plan on presenting these images at the Jamboree.

We performed our novel PHB extraction method on our E. coli expressing the pha plasmid to quantify PHB yield. Through sonication and separation by density differentials our team extracted 0.0427g of PHB.

**PHB extraction with sonication and differential centrifugation**

Our team created a unique and environmentally conscious method for PHB extraction free of potent chemicals. Typical extraction methods involve chloroform, methanol, and sodium dodecyl sulfate. Not only are these chemicals potentially toxic to humans if exposed in high quantities. The proper laboratory disposal of chemicals such as chloroform and methanol that are considered “hazardous waste” prove harmful to the environment. Chloroform has a long retention time in the atmosphere and can prove harmful to humans if not properly disposed. This causes a strain on the need for safe disposal and isn’t effective for long term production of PHB.3 Our novel method for PHB extraction uses sonication and sucrose as its main essential components, thus reducing the environmental impact of extraction. This protocol is detailed in our protocol handbook.This protocol is still undergoing empirical trials to improve PHB yield and purity, but our team was able to create a novel method for PHB extraction with the environmental focus in line with our project.

![](https://static.igem.org/mediawiki/2019/4/48/T--Virginia--2019_epi.jpg)![](https://static.igem.org/mediawiki/2019/1/17/T--Virginia--2019_pellet.jpg)

(Left) PHB pellet (bottom pellet) and cell pellet (top pellet) separated by a sucrose cushion of 1.23 g/mL. (Right) Dried PHB pellet obtained from bottom of density differential centrifugation (mass = .0427 g).

**Additional Characterization of BBa\_K934001**

Our team performed a preliminary Red Nile staining of the cultures after 72 hour culture of K12 DH5 E. coli at 37°C in M9 minimal medium. The choice of M9 as opposed to LB used by Tokyo Tech was due to the ideal nitrogen limiting conditions of the medium, which is a proven factor to influence PHB production1. 72 hours was also cited as the culture time for maximum PHB/cell yield.

Our team also wanted visual insight of PHB production in a cell on the cellular level. With the help of the University of Virginia Medical School Microscopy Center our team gather Electron Microscopy images of the production of PHB by cells expressing BBa\_K934001. Due to time constraints our EM pictures were not obtained in time for upload to our wiki, but we plan on presenting these images at the Jamboree.

To characterize and quantify the production of PHBs from part BBa\_K934001 our team performed Red Nile staining to determine if PHBs were produced under these conditions. As can be seen by Red Nile results from a non PHB producing control and the BBa\_K934001 expressing cells, PHBs were indeed produced through 72 hour cultures in M9 minimal medium.

![](https://static.igem.org/mediawiki/2019/2/2e/T--Virginia--2019_dots_1.jpg)![](https://static.igem.org/mediawiki/2019/2/2e/T--Virginia--2019_dots_2.png)

Red-nile procedures done on a PHA producing bacteria using part BBa\_K934001 expressed in E. coli DH5-alpha(left) and DH5-alpha transformed with a chloramphenicol resistance backbone as a control (right).

## Summary

Although our team was not able to fully characterize the level of functionality of our device, significant improvements were made in many aspects of our project. Our team was successful in developing a plasmid that was able to metabolize styrene to an extent while also producing PHB.This features marks one of the essential BioBricks to creating our styrene degrading and PHB producing device. Quantitative and qualitative data gathered on PHB production of our plasmid indicated PHBs produced at comparable amounts to literature, rendering this half of our device both functional and effective. Although our team was not able to perform extensive testing, characterization of the functionality of our *sty* plasmid indicates that it is able to sustain cells expressing it in growth medium with styrene as the sole carbon source. Further characterization to determine optimal levels of styrene integration and to quantify the metabolic rate at which styrene is Our custom synthetic ribosomal binding sites provide a toolbox that we hope future teams will utilize to increase the efficiency in the context of their own unique biological devices. Additionally, we concluded that the introduction of this new T7 promoter does not require T7 polymerase for transcription, and that regular polymerase is sufficient to allow transcription. This promoter that solely relies on the presence of IPTG for high efficiency transcription allow tight regulation of expression of our devices.

In the next two weeks leading up to the Jamboree, our team is actively working to continue characterization, quantification and confirmation of our entire device in a single chassis that expresses both the *sty* and *pha* plasmid, and is capable of metabolizing styrene as its sole carbon source to produce polyhydroxybutyrate.

1. Ward, P. G., Roo, G. de & O’Connor, K. E. Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3. Appl. Environ. Microbiol. 71, 2046–2052 (2005).
2. Spiekermann, P., Rehm, B. H. A., Kalscheuer, R., Baumeister, D. & Steinbüchel, A. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds.
3. Chloroform. Scottish Pollutant Release Inventory

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