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Team:Purdue/Project
From 2008.igem.org
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=== Part 1: Lit Research === | === Part 1: Lit Research === | ||
| - | + | See the Resources page for a brief list of papers. | |
| - | + | As for any research project, we started by reading...and reading...and reading some more. We determined that our project was feasible. Modeling has been done of the SOS and phr pathways, using UV radiation to trigger them. Normally, however, the promoters of these pathways were linked to GFP or other fluorescent proteins. As a real-time biosensor, fluorescence was not really an option. We wanted people to be able to see the colors change as it happens--while they're still in the sun. After figuring out which genes we wanted to use, we looked in the Registry--and found them! | |
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After combing the Registry of Standard Biological Parts, we found 2 parts that we could use to implement our idea. First, part J22106 (contributed by Bangalore in 2006) is the promoter for ''recA'', a central gene in the bacterial SOS pathway. Next, we found a complete ''lacZ'' (I732017) which could be attached to the promoter. Both parts are relatively OK according to the quality control tests. By cloning the sequence of promoter-reporter, we can make the traditional if-then construct often used to test promoter strength. In this case, however, we will clone it into ''lac-'' cells (so we don't get false positives). By plating on X-gal plates, those cells that have successfully transformed will turn blue. | After combing the Registry of Standard Biological Parts, we found 2 parts that we could use to implement our idea. First, part J22106 (contributed by Bangalore in 2006) is the promoter for ''recA'', a central gene in the bacterial SOS pathway. Next, we found a complete ''lacZ'' (I732017) which could be attached to the promoter. Both parts are relatively OK according to the quality control tests. By cloning the sequence of promoter-reporter, we can make the traditional if-then construct often used to test promoter strength. In this case, however, we will clone it into ''lac-'' cells (so we don't get false positives). By plating on X-gal plates, those cells that have successfully transformed will turn blue. | ||
| - | Standard Assembly methods were used. Stabs of transformed cells containing each part were obtained from iGEM. Next, a miniprep was done for each part, and each part was digested. | + | Standard Assembly methods were used. Stabs of transformed cells containing each part were obtained from iGEM. Next, a miniprep was done for each part (using QIAGEN miniprep kits for microcentrifuge), and each part was digested using restriction enzymes and buffers from New England Biolabs. To make sure the recA promoter was in front of lacZ, we first cut the SOS plasmid with EcoRI and SpeI, which left us the promoter all by itself, a 192 bp part. We cut lacZ with EcoRI and XbaI, which cut out the piece of plasmid in front of lacZ, leaving a part about 5000 bp long. After digestion, we ran our parts through an agarose gel, and purified the bands of the correct sizes (using a QIAGEN QIAquick Gel Extraction Kit). Next in the process was ligation, again using materials from New England Biolabs. Finally, the new plasmids were transformed into chemically competent DH5a cells. |
| + | |||
| + | Testing of the new bacteria to follow, as well as submission of the new part to the Registry. | ||
| + | |||
| + | For more detailed protocols, see the Notebook page. | ||
Revision as of 16:38, 9 October 2008
Contents |
Overall project
This year at Purdue, our goal is to make a bacterial UV sensor for commercial application. By exploiting existing E. coli DNA repair pathways (photoreactivation and SOS); we want to eventually create a "patch" that will change colors as UV exposure increases. Thus, one would be able to test when new sunscreen needs to be applied based on actual DNA damage. Other applications could include Bacterial "tattoos" that only show up in the sun, color-changing T-shirts, etc.
Biologically, we are planning to attach the phr (photoreactivation) promoter to a gene creating some kind of red color, such as RFP or prodigiosin or LacYZ on MacConkey agar. As a result, as pyrimidine dimers are formed, the natural photoreactivation pathway will be activated by the bacteria and red color will develop alongside natural DNA repair. Once more severe DNA damage occurs, the E. coli will naturally switch over to the well-documented SOS (recA) pathway. By combining the promoter for this pathway (a part used by Bangalore in 2006) with the lacZ gene, severe UV damage will make beta-galactosidase which will cleave X-gal which will create a blue pigment. Thus, our device will slowly turn red and eventually blue as the DNA damage resulting from UV radiation increases.
Project Details
Unfortunately, there is insufficient documentation regarding the photoreactivation pathway. Because this pathway is not present in humans, very little research has been done on the subject. As a result, there is no definitive source for the specific genetic code that makes up the promoter of the system. Because of this and other funding problems, the Purdue team has decided to focus on just the SOS side of the project.
Part 1: Lit Research
See the Resources page for a brief list of papers. As for any research project, we started by reading...and reading...and reading some more. We determined that our project was feasible. Modeling has been done of the SOS and phr pathways, using UV radiation to trigger them. Normally, however, the promoters of these pathways were linked to GFP or other fluorescent proteins. As a real-time biosensor, fluorescence was not really an option. We wanted people to be able to see the colors change as it happens--while they're still in the sun. After figuring out which genes we wanted to use, we looked in the Registry--and found them!
Part 2: Modeling
As the Purdue team consists of mostly engineers, it is our goal to be able to mathematically model our system. A working model will help us understand the mechanisms involved in our genetic modifications, and will allow us to predict the consequences of any modifications.
For more details, see the Modeling page.
Part 3: In the Lab
After combing the Registry of Standard Biological Parts, we found 2 parts that we could use to implement our idea. First, part J22106 (contributed by Bangalore in 2006) is the promoter for recA, a central gene in the bacterial SOS pathway. Next, we found a complete lacZ (I732017) which could be attached to the promoter. Both parts are relatively OK according to the quality control tests. By cloning the sequence of promoter-reporter, we can make the traditional if-then construct often used to test promoter strength. In this case, however, we will clone it into lac- cells (so we don't get false positives). By plating on X-gal plates, those cells that have successfully transformed will turn blue.
Standard Assembly methods were used. Stabs of transformed cells containing each part were obtained from iGEM. Next, a miniprep was done for each part (using QIAGEN miniprep kits for microcentrifuge), and each part was digested using restriction enzymes and buffers from New England Biolabs. To make sure the recA promoter was in front of lacZ, we first cut the SOS plasmid with EcoRI and SpeI, which left us the promoter all by itself, a 192 bp part. We cut lacZ with EcoRI and XbaI, which cut out the piece of plasmid in front of lacZ, leaving a part about 5000 bp long. After digestion, we ran our parts through an agarose gel, and purified the bands of the correct sizes (using a QIAGEN QIAquick Gel Extraction Kit). Next in the process was ligation, again using materials from New England Biolabs. Finally, the new plasmids were transformed into chemically competent DH5a cells.
Testing of the new bacteria to follow, as well as submission of the new part to the Registry.
For more detailed protocols, see the Notebook page.
Part 4: Results
No results yet! See the Modeling page for expected results...
Failed ideas???/Future Plans
- Also include ppt here?
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