Team:Virginia/results
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.
sty Plasmid Characterization
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.
PHB Production
Pha plasmid induced by IPTG
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.
(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.
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.
- 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).
- 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.
- Chloroform. Scottish Pollutant Release Inventory
