Difference between revisions of "Team:Lambert GA/Demonstrate"

A critical component of characterization and comprehension of synthetic biology, software, and hardware tools exists in the form of mathematical modeling. Here we outline the models developed to facilitate the understanding of CALM (machine learning model) and ElectroPen (ultralow-cost synthetic biology tool), and assist in the prevention of cholera outbreaks. The model for CALM serves to develop a proactive response to potential outbreaks by utilizing predictive algorithms to identify at-risk areas and inform relevant organizations to mount a response. The model for the ElectroPen demonstrates the underlying principles allowing for the functionality of this ultralow-cost electroporator in terms of mechanical, electrical, and biological engineering. With the ElectroPen facilitating infield biosensor tests and CALM predicting cholera outbreaks, the described mathematical modeling serves to describe two key elements of CAPTIVATE and the mechanisms behind their performance.

In order to utilize LacZ color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the LacZ operon is induced and subsequently breaks down Xgal and produces galactose and a blue pigment. This color can then be characterized by the shade of the blue pigment expression. This part is transformed in pSB3C5 instead of pSB1C3 because when LacZ is induced in a high copy plasmid such as pSB1C3, it drains the metabolism of a cell.

Lambert iGEM used sequences and DNA shared from the Styczynski Lab at the Georgia Institute of Technology to build a T7, Toehold, LacZ switch to be applied as a biosensor. We tested the Biobricked T7 Toehold LacZ Switch BBa_K2550000, the Biobricked T7 Trigger Sequence BBa_K2550001 T7 Toehold LacZ Switch obtained from GATech, and the pSB6A1 T7 Trigger on Luria broth plates with chloramphenicol, carbenicillin, and Xgal antibiotic resistance.



The dual plasmid transformation using the pSB6A1 trigger and biobricked toehold was successful. Since the blue expression was only evident on the dual plasmid plate and not on the plate that contained the toehold, the trigger is successful in inducing LacZ gene expression.

The transformations of the biobricked toehold construct were unsuccessful because they produced a blue color. The blue might be a result of the orthogonal switch not binding as expected or a result of overexpression from a too strong promoter. Since the trigger sequence was not transformed with the construct, the colonies should appear white as the toehold only reveals the RBS and starting sequence of LacZ when the trigger sequence. This could be due to a leaky expression of LacZ under the strong T7 promoter. The dual plasmid transformation confirmed that neither the biobricked trigger nor the biobricked toehold sequence worked in conjunction with each other although they worked in conjunction with similar parts not in pSB1C3.






We aligned the sequencing results to the original trigger sequence obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. Since the sequences matched after alignment, this confirmed that the trigger sequence was correctly cloned.

In recognition of the dire crisis cholera outbreaks have presented to the world, inflicting harm on millions and killing hundreds of thousands, we present a new solution to ensure a proactive response to cholera outbreaks can be mounted by targeting contamination within the water sources. CAPTIVATE constitutes a portable diagnostic kit to test the water in local areas to determine whether Vibrio Cholerae is present or not. Cholera diagnosis requires This diagnostic kit includes Lambert iGEM’s hardware components such as the 3-D Fuge, Chrome-Q, Chrome-Q Base, and the Electropen System. The kit also contains the wet-lab components including the Spira Swab (gauze water filter), freeze-dried Biosensor cells in culture tubes, and a RNA extraction kit. The kit is capable of powering a phone for the Color-Q app and a cooling system needed for certain wet-lab parts of the project through a solar panel. CAPTIVATE portable diagnostic kit could work in conjunction of existing World Health Organization treatment kits which can provide a more efficient assessment to determine if cholera exists in that water source.

Materials

• Scoop (collection jar-disposable water bottle)
• Muslin (Muslin will be placed in the 100 mL Nalgene bottle filled with diH2O: This diH2O can be obtained from boiling the water when sterilizing the muslin cloths).
• 100 ml Nalgene bottle with 2cm.hole at the bottom (will be wrapped around with aluminum foil to keep sterile)
• Sterile 100 mL Nalgene bottle
• Crochet Hook, or other straight stick that can be sterilized and reused
• lysozyme in TE buffer
• proteinase K
• Rneasy Mini Kit
• 1M HCl
• 10 ml sterile distilled H2O (1ml needed for spin columns, and disposal of biosensor cells)
• Electropen System (Cuvette and Electropen)
• X-gal
• Freeze-Dried Cells in a vial
• LB in vials
• Culture Tubes
• Microcentrifuge Tubes
• PCR Tubes
• 50 ml conical tubes
• Lego spectrophotometer
• 1% sodium hydrochlorite
• 10% glycerol
• Chrome-Q Dome & Chrome-Q base
• Color-Q app and phone connected to the solar panel.
• 3-D fuge
• pipette/micropipette