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The Biohacker Kit
The premise of this project was to create a linked two plasmid system to create a bio-system whose inputs and outputs can be easily swapped. The purpose of this system is to create a means by which students can be easily introduced to synthetic biology (the design and re-design of biological systems for useful purposes) in their classrooms without the need for high-tech, expensive equipment.
How It Works
The system uses two different types of plasmids (pACYC184 as the input plasmid and pBR322 as the output plasmid). The input plasmid had the promoter gene along with a phage activator ligated into it, and the reporter promoter had a phage promoter and a reporter gene ligated into it. When the promoter is activated, it will trigger the phage activator. The phage activator will then activate the phage promoter in the output plasmid, which will turn on the output gene. In this way, there could be a whole palate of input and output plasmids that can be mixed and matched at the students' will to easily create their own individual systems. The two plasmids have different antibiotic resistances so that one can tell when their programmed plasmids have been successfully transformed into cells, along with different origins of replication. Using two different antibiotic resistances will help ensure that the colonies have both plasmids co-transformed inside the E. coli cells.
Figure 1: Two plasmid system, using the UV promoter as an input and GFP as the output
The Biohacker Kit would be a box that schools could buy that contained all of the input and output constructs. The lab work involved would be to co-transform the input and output of the student's choice with E. coli cells, and plate them on the correct antibiotic plates. After overnight incubation, the students can see all of the colonies and then activate the promoter to kick-start the system. Then, depending on the promoter, over the next day or two the system will begin to express the reporter gene! Students will also have the ability to monitor the system to see how the gene expression varies with time.
Results
Our composite part (BBa_K1354000) uses the sulA promoter (biobricked by UT-Tokyo in 2011 - BBa_K518010), and the delta activator from phiR73 phage (biobricked by Cambridge in 2007 - BBa_I746352). The phage activator turned on a phage promoter with a GFP reporter composite part,
| LB Control | LB UV | UV Control | UV UV | GFP Control | GFP UV | Joint Contorl 1 | Joint Control 2 | Joint Control 3 | UV + GFP 1 | UV + GFP 2 | UV + GFP 3 | |
| Concentration (OD600) | 0.802 | 0.186 | 0.713 | 0.357 | 0.733 | 0.289 | 0.406 | 0.469 | 0.566 | 0.103 | 0.346 | 0.141 |
| GFP (485/520nm) | 1788 | 1065 | 1686 | 1273 | 2153 | 1274 | 14138 | 13169 | 20566 | 6216 | 26585 | 9659 |
| Normalized GFP | 2229 | 5725 | 2364 | 3565 | 2937 | 4408 | 34822 | 28078 | 36335 | 60349 | 76835 | 68503 |
Figure 1: Fluorescence Intensity for Three Colonies of UV+GFP and Controls
Future Work
We built our system with restriction sites around the UV promoter and GFP reporter so that they can be cut out and replaced with other inputs and outputs easily. Going forward, we hope to create an entire kit, consisting of at least four more inputs and four more outputs. Any input or output can be used from the BioBrick registry, as long as primers are designed to incorporate the correct restriction enzyme sites.
Figure 2: The duel plasmid reporter system, demonstrating variable input/output options
References
- Chang AC, Cohen SN. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134(3):1141-56
- Paulina Balbás, Xavier Soberón, Enrique Merino, Mario Zurita, Hilda Lomeli, Fernando Valle, Noemi Flores, Francisco Bolivar. (1988) Plasmid vector pBR322 and its special-purpose derivatives — a review.Gene 50(1–3): 3-40
