Team:Purdue/Project

From 2008.igem.org

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, 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 and plating on X-gal, 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 phr 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 region 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/Background

See the Resources page for a brief list of references.

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 activate 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!

For some background, lacZ has been used for blue/white screening of E. coli for decades. Standard E. coli naturally make the lacZ protein (beta-galactosidase, or beta-gal), which reacts with X-gal, a structurally relative chemical to lactose (which is cleaved by beta-gal into monosaccharides). However, when beta-gal cleaves X-gal, one of the chemical components formed is a bright blue pigment, 5-bromo-4-chloro-3-hydroxyindole. Therefore, normal E. coli, when plated on X-gal with IPTG (an inducer of lacZ) turn blue. If a lac- E. coli is transformed with a part containing either the alpha constituent of lacZ or the entire gene, those that transform successfully will turn blue, while the rest (which are still lac-) will be white. This process can also be done in reverse, where the new gene will splice the coding region responsible for lacZ, making it ineffective. Thus, the successful bacteria will be white (and everything else will be blue). For this project, we are attaching a different promoter to the front of lacZ, as opposed to the normal operon. Thus, lacZ will be transcribed at the same time as the system corresponding to the promoter is transcribed. Therefore, as our promoter is activated, the E. coli will turn blue.

When DNA damage occurs, kinks and gaps are formed in the DNA strand. Thus, single-stranded DNA (ssDNA) is formed when the cell attempts to transcribe the kinked DNA. In particular, UV-B radiation forms thymine-thymine (cyclopyrimidine) dimers between adjacent thymines. The cell will first activate two initial DNA repair systems. First, the phr pathway will specifically fix thymine dimers with the help of visible light. This process occurs constantly at very low levels in cells. If there is too much ssDNA for phr to keep up, the cell activates nucleotide excision repair (NER), which effectively eliminates most of the damage with very high accuracy. NER is considered the first phase of the SOS pathway, as its genes (uvrABD) are normally inhibited by the lexA repressor (a characteristic of all ~40 SOS genes). However, if DNA damage is extreme, other SOS genes, including recA, are activated as a last resort. This next set of genes causes the cell to form random mutations via DNA Polymerase V (a mutagenic polymerase) which will add any base into a gap, as opposed to the correct one. For this project, we are using the recA promoter, which is a standard promoter of the SOS pathway and is activated only after extreme DNA damage.

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 (recA) 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

We made the part, and sent it in! See the Notebook/Project page, or the powerpoint/poster below for more details. Unfortunately, due to lack of funding and time, we were unable to test our part. For the expected results, see the Modeling page.

Part 5: Safety Considerations

Our project is intended to raise the public's awareness of the lack of danger posed by bacterial mutations. Our eventual hope would be that an application involving our living biosensor would be possible. Our cloned bacteria are already engineered so that they cannot survive outside of a lab environment. In other words, our protocol was entirely under Biosafety Level I (i.e., little or no health hazard).

At Purdue, the group in charge of recombinant DNA research is the Institutional Biosafety Committee (IBC). In cooperation with them, we filled out a form detailing our entire protocol. We then had to indicate any health hazards that could be posed by our project (which there were none).

Failed Ideas

In order to make a multicolored sensor, we would first need to obtain DNA coding for the promoters of uvrA (the first gene activated in NER) or phr. Unfortunately, a complete analysis of the promoters of phr are not available, and uvrA is not part of the Registry, so we do not have access to it currently. Ideally, the phr pathway would be preferable for this application, as NER will fix any DNA damage, whereas phr focuses specifically on the dimers formed by UV-B radiation.

Other than the lacZ/X-gal reporter combination, other options were explored. Initially, we were attracted to the red color of Lycopene (the red pigment in tomatoes), a gene put in the Registry by Edinburgh. Unfortunately, this part cannot effectively produce any visible pigment in a timely manner. The next option we looked at was MacConkey agar, which works similarly to X-gal: a lactose-fermenting E. coli will turn red on MacConkey agar. Therefore, if supplied with lactose and a lacZ gene, lac- E. coli would change colors if attached to the correct promoter. Unfortunately, this system would require us to create 2 different strains of bacteria (which would have to be kept separate) because they would both be using the lacZ gene. The next possibility for a color promoter was an RFP gene. RFP is distinctive from other fluorescent proteins because it turns bacteria a visible red color. Upon further study, both standard RFP and the its successor mCherry require several hours to be produced to an extent to make them visible without fluorescence. Again, this timescale is too long for our application. Our last idea for a visible reporter was the prodigiosin pigment, a pigment naturally made in several strains of bacteria. When constitutively produced, prodigiosin appears blood-like: bright red and shiny. Unfortunately, the prodigiosin pathway also requires more than 20 separate genes, all of which would have to be transformed into E. coli. This is not very feasible for this project.

Future Plans

Upon successfully creating an SOS biosensor, we would like to continue the project in several ways. First, we would modify the system to improve the speed of color changing. This could be accomplished by creating an NER- mutant, which would necessarily skip over NER and activate the SOS system almost immediately following any significant UV damage. As stated before, we would like to add a secondary color system which would probably utilize the uvrA gene of the NER pathway. Another goal of this team would be to adapt the biosensor to a marketable device, such as a wearable patch. This way, the true effects of sunscreen could be tested: the patch would change colors as the sunscreen wore off and the DNA was damaged.

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