Team:Cadets2Vets/Design

Cadets2Vets utilized literature searches and the iGEM Registry of Standard Biological Parts to identify the appropriate parts and sequences to use for the arsenic circuit. We knew we needed to have unregulated expression of ArsR and that ArsR needed to control expression of the GFP reporter. We found constitutive and regulated promoters to fit our needs.

Then we used DNA sequence analysis software, like Genome Compiler, to assemble a virtual sequence of what we needed. We used this software to add in additional features like the BioBrick Prefix and Suffix, as well as make predictions regarding the cloning steps we would take to create the arsenic circuit. The sequences of the Parts we selected can be found here.

To test the plasmids, we would use a variety of assays that would assess whether the correct proteins were being generated and whether the proteins were functioning as expected.

The creation of the circuit was achieved by obtaining synthesized DNA from Integrated DNA Technologies and using enzymes and competent E. coli cells obtained from New England Biolabs’ BioBrick Assembly Kit. DNA was cut using restriction enzymes and then linked to a vector backbone or to another piece of DNA using T4 ligase. The plasmid was then transformed into bacteria and then the plasmid DNA was isolated. We screened for the correct plasmid by using restriction enzyme digest and seeing that the resulting fragment matched our predictions.

In general, the strategy was to:

determine that proteins of the correct size were being produced
determine that the GFP was fluorescing when we expected it to fluoresce
optimize the assay conditions for the in vitro transcription/translation protocol
optimize transfer of the assay to paper tickets.

Our initial assessments of the plasmids were performed by monitoring the effect of a range of arsenic concentrations on the arsenic circuit being expressed by live E. coli cells, examining GFP fluorescence using a Light Cycler as a spectrophotometer, and looking at protein expression using a protein gel.

As we move forward in testing our circuit, we hope to identify the concentration range of arsenic that our circuit can report; the working conditions that the circuit will function, such as temperature and incubation time of the assay; and the specificity of the circuit. Additionally, our team is developing a portable imaging system using technology made by ECBC.

The use of cell-free extracts is important to the progress and future development of the arsenic circuit, and other similarly styled circuits.

The first advantage is that the use of cell-free extracts increases the shelf-life of the assay. We expect to be able to lyophilize the enzymes and proteins used in the arsenic circuit, so we do not have to be concerned with keeping bacteria cells alive and healthy.

The second advantage is that there will be no chance of accidentally releasing a modified bacteria to the environment. Although there is a low risk that the bacteria we use would adversely affect the environment, due to the required selective pressure needed to maintain the plasmid and the relative safety of the produced proteins, we will maintain laboratory safety standards and laws that prohibit the release of genetically modified organisms.

The third advantage is that we expect to be able to detect a broader range of arsenic using a cell-free system. Using live cells requires arsenic to be able to transport into the cell and interact with the necessary proteins. Having the proteins readily available in solution should reduce the barrier of arsenic accessing ArsR.

Team:Cadets2Vets/Design

DESIGN

Our circuit utilizes an arsenic regulatory protein (Arsr) and the green fluorescent protein (GFP) gene so that when Plasmid-containing cells are exposed to arsenic ions, gfp is produced, making the ticket glow under a blue or ultraviolet light.

the Thought Process

design

build

test

cell-free lysate

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