Team:Minnesota/Project/UV Absorption

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<h1>Synthesizing UV-Protective Compounds in Bacteria</h1><br>
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<p>Our first idea was to engineer the production of the Micosprine-like Amino Acids (MAAs) in <i>E. coli</i>. These compounds confer UV protection in Anabaena variabilis and are currently used in sunscreen that protects against the entire UVA and UVB spectrum. To obtain these compounds for use in industry, the current method is direct extraction from A. variabilis. To develop an alternate way of producing these compounds, genes coding for enzymes in their production pathways were taken from A. variabilis and cloned into <i>E. coli</i> using BioBrickTM techniques. The result: E. coli that can produce UV protectant compounds, which has several implications. E. coli are cheap, easy to produce, grow quickly, are preferred for use in industry, and can be grown up in giant bioreactors. Therefore the speed and ease at which MAA-producing bacteria can be grown, as well as the amount that can be grown would drastically increase. Additionally, promoters and mechanisms of down-regulation in <i>E. coli</i> are well characterized, making modification and control of the pathway much simpler. These parts could easily be employed in future experiments where increased UV tolerance is desired (for example, in the characterization of a UV-sensitive promoter).  Alternatively, a skin-based, UV-protectant probiotic could eventually be developed if these parts are incorporated into a native, non-pathogenic skim bacterium chassis, such as <i>Staphylococcus epidermidis</i>.  Imagine a “sunscreen” that grows naturally on your skin!</p>
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<p><b>Goal</b><br>
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Clone multiple gene pathways from algal species into <i>E. coli</i> to produce natural UV-protective sunscreening compounds.</p>
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<p><b>Background</b><br>
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Our research has focused on two novel biosynthetic pathways found in two distinct algal species. A pathway ending in the production of two UV-protective compounds, shinorine and mycosporine-glycine, was cloned from <i>Anabaena varibalis</i>. A second pathway leading to the production of the unrelated UV-screening compound scytonemin was cloned from <i>Nostoc punctiforme</i>. Our objective is to develop novel and effective production platforms for these compounds, some of which are currently used in expensive sunscreens and lotions. These compounds are used both for the UV-absorptive properties as well as their role as potent antioxidants.</p>
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/1Bac.png">
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<p><b>Figure 1. Biosynthetic pathways of MAAs.</b> a) Shinorine biosynthesis.  DHQS, dehydroquinate synthase; O-MT, O-methyltransferase; ATP-grasp; NRPS, non-ribosomal peptide synthase.  b) Scytonemin biosynthesis.  ScyA (NpR1276), acetolactate synthetase; ScyB (NpR1275), leucine dehydrogenase; ScyC (NpR1274), protein of unknown function.</p>
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<p><b>Methods</b><br>
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<p>Genomic DNA was obtained from the Brett Barney Lab (University of Minnesota) and ATCC for <i> A. variabilis</i> and <i>N. punctiforme</i>, respectively. From here, PCR primer extension was used to clone each individual gene (seven in total) with flanking restriction digest sites to be used for vector cloning. Each gene was cloned into an individual pUCBB BioBrick™ cloning vectors. From here, genes from each individual pathway were cloned onto one pUCBB BioBrick™ vector for each specific pathway.</p>
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<p>Upon successful cloning of each pathway, high-performance liquid chromatography (HPLC) was used to analyze production of mycosporine-glycine, shinorine, and scytonemin. Methods for extraction and analysis of these compounds from liquid E. coli cultures have previously been developed by Balskus and Walsh (2011).</p>
 +
 
 +
<p>Additionally, UV-sensitivity screens were performed on E. coli cultures expressing target pathways. A preliminary screen was done using different exposure time points (t=5, 10, 15, 20 minutes) and dilutions. A secondary test was performed with a 104 fold dilution and an exposure time of 20 minutes.  Treated cells were plated and then CFUs were counted.</p>
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<p><b>Parts List</b><br>
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BBa_K814000 &nbsp;&nbsp;&nbsp;  dehydroquinate synthase (DHQS) generator <br>
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BBa_K814001 &nbsp;&nbsp;&nbsp; ATP-grasp (ATPG) generator <br>
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BBa_K814002 &nbsp;&nbsp;&nbsp; dehydroquinate O-methyltrasferase (O-MT) generator <br>
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BBa_K814003 &nbsp;&nbsp;&nbsp; shinorine non-ribosomal peptide synthase (NRPS) generator <br>
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BBa_K814004 &nbsp;&nbsp;&nbsp; mycosporine-glycine biosynthetic pathway <br>
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BBa_K814005 &nbsp;&nbsp;&nbsp; shinorine biosynthetic pathway <br>
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BBa_K814006 &nbsp;&nbsp;&nbsp; negative control for mycosporine-like amino acid biosynthesis <br>
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BBa_K814007 &nbsp;&nbsp;&nbsp; ScyA (acetolactate synthase) generator <br>
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BBa_K814008 &nbsp;&nbsp;&nbsp; ScyB (leucine dehydrogenase) generator <br>
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BBa_K814009 &nbsp;&nbsp;&nbsp; ScyC generator <br>
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BBa_K814010 &nbsp;&nbsp;&nbsp; partial scytonemin biosynthetic pathway, scyCB <br>
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BBa_K814011 &nbsp;&nbsp;&nbsp; scytonemin biosynthetic pathway, scyBAC <br></p>
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<p><b>Results</b><br>
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/2_Bac.png">
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<p><b>Figure 2.  HPLC analysis of E. coli expressing MAA biosynthetic genes.</b> Cell extracts of E. coli transformed with +DOA (DHQS, O-methyl transferase, and ATP-grasp) and +DOAN (DOA, and NRPS) showed production and 4-deoxygadusol (a) and mycosporine-glycine (b). Shinorine was not detected in the +DOAN expressing cells. Samples were compared against a +DA negative control (this construct lacks the ability to produce the 4-deoxygadusol intermediate). Cells expressing +ABC (ScyA, ScyB, ScyC) did not show scytonemin production. Data was compared with previous results from Balskus and Walsh, as a standard could not be obtained. </p>
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/3Bac.png">
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<p><b>Figure 3. UV sensitivity screens of E. coli cultures producing MAAs.</b>  Preliminary results for the secondary screen showed increased survival in cells producing mycosporine-glycine when compared with +DO and +DA negative controls. Cells expressing +ABC did not show increased survivability.</p>
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/cellimages.jpg">
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<h1>UV Absorption by Skin Microbes</h1><br>
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<p><b>Figure 4.  Visual inspection of E. coli cells containing the scytonemin pathway.</b> Although HPLC and growth tests were negative, cells expressing +ABC were seen to be expressing a deep brown product that could easily be visualized (below). The control is a cell culture expressing a +DA plasmid vector. </p>
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<p><b>Conclusion</b><br>
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Successful transformation and expression of the mycosporine-glycine/shinorine pathway was achieved, and production of mycosporine-glycine was observed via HPLC analysis. This appears to incur resistance to UV-radiation to a certain degree. As we move forward, it will become critical to obtain expression of NRPS, the final gene in this pathway, and production of shinorine. Steps are being taken to optimize of this final step in the biosynthetic pathway is ongoing.</p>
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Between 2 and 3 million cases of nonmelanoma skin cancers arise each year, making skin cancer the most prevalent form of cancer worldwide. With skin cancer such a prominent problem worldwide, we are looking into alternative methods to protect against UV radiation. Photosynthetic organisms create an array of compounds, called mycosporine like amino acids, which naturally protect them against UV radiation. The cyanobacterium, Anabaena variabilis, contains a cluster of four genes capable of producing two UV protective compounds, mycosporine-glycine and shinorine.  Utilizing BioBrick™ vector techniques, we intend to clone these genes out of A. variabilis and into Escherichia coli for characterization.  After characterization and determination of the efficiency of the genes in E. coli, we will clone the shinorine gene cluster into Staphylococcus epidermidis. Successful introduction of this gene cluster and expression of UV-protective compounds could be useful as a one-time-application alternative to currently marketed sunscreens.  
+
<p>Although conclusive evidence regarding the production of scytonemin was not found, the initial results are promising. Previous studies on algae described a deep brown secretion in cells that were shown to be producing this compound. This pathway has not yet been fully characterized, and additional genes with unknown functions are present within the native <i>N. punctiforme</i> gene cluster. We are currently looking into cloning and expressing these additional genes, and looking for ways to isolate and analyze potential intermediate products within this pathway.</p>
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Latest revision as of 03:58, 4 October 2012

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Synthesizing UV-Protective Compounds in Bacteria


Our first idea was to engineer the production of the Micosprine-like Amino Acids (MAAs) in E. coli. These compounds confer UV protection in Anabaena variabilis and are currently used in sunscreen that protects against the entire UVA and UVB spectrum. To obtain these compounds for use in industry, the current method is direct extraction from A. variabilis. To develop an alternate way of producing these compounds, genes coding for enzymes in their production pathways were taken from A. variabilis and cloned into E. coli using BioBrickTM techniques. The result: E. coli that can produce UV protectant compounds, which has several implications. E. coli are cheap, easy to produce, grow quickly, are preferred for use in industry, and can be grown up in giant bioreactors. Therefore the speed and ease at which MAA-producing bacteria can be grown, as well as the amount that can be grown would drastically increase. Additionally, promoters and mechanisms of down-regulation in E. coli are well characterized, making modification and control of the pathway much simpler. These parts could easily be employed in future experiments where increased UV tolerance is desired (for example, in the characterization of a UV-sensitive promoter). Alternatively, a skin-based, UV-protectant probiotic could eventually be developed if these parts are incorporated into a native, non-pathogenic skim bacterium chassis, such as Staphylococcus epidermidis. Imagine a “sunscreen” that grows naturally on your skin!

Goal
Clone multiple gene pathways from algal species into E. coli to produce natural UV-protective sunscreening compounds.

Background
Our research has focused on two novel biosynthetic pathways found in two distinct algal species. A pathway ending in the production of two UV-protective compounds, shinorine and mycosporine-glycine, was cloned from Anabaena varibalis. A second pathway leading to the production of the unrelated UV-screening compound scytonemin was cloned from Nostoc punctiforme. Our objective is to develop novel and effective production platforms for these compounds, some of which are currently used in expensive sunscreens and lotions. These compounds are used both for the UV-absorptive properties as well as their role as potent antioxidants.

Figure 1. Biosynthetic pathways of MAAs. a) Shinorine biosynthesis. DHQS, dehydroquinate synthase; O-MT, O-methyltransferase; ATP-grasp; NRPS, non-ribosomal peptide synthase. b) Scytonemin biosynthesis. ScyA (NpR1276), acetolactate synthetase; ScyB (NpR1275), leucine dehydrogenase; ScyC (NpR1274), protein of unknown function.

Methods

Genomic DNA was obtained from the Brett Barney Lab (University of Minnesota) and ATCC for A. variabilis and N. punctiforme, respectively. From here, PCR primer extension was used to clone each individual gene (seven in total) with flanking restriction digest sites to be used for vector cloning. Each gene was cloned into an individual pUCBB BioBrick™ cloning vectors. From here, genes from each individual pathway were cloned onto one pUCBB BioBrick™ vector for each specific pathway.

Upon successful cloning of each pathway, high-performance liquid chromatography (HPLC) was used to analyze production of mycosporine-glycine, shinorine, and scytonemin. Methods for extraction and analysis of these compounds from liquid E. coli cultures have previously been developed by Balskus and Walsh (2011).

Additionally, UV-sensitivity screens were performed on E. coli cultures expressing target pathways. A preliminary screen was done using different exposure time points (t=5, 10, 15, 20 minutes) and dilutions. A secondary test was performed with a 104 fold dilution and an exposure time of 20 minutes. Treated cells were plated and then CFUs were counted.

Parts List
BBa_K814000     dehydroquinate synthase (DHQS) generator
BBa_K814001     ATP-grasp (ATPG) generator
BBa_K814002     dehydroquinate O-methyltrasferase (O-MT) generator
BBa_K814003     shinorine non-ribosomal peptide synthase (NRPS) generator
BBa_K814004     mycosporine-glycine biosynthetic pathway
BBa_K814005     shinorine biosynthetic pathway
BBa_K814006     negative control for mycosporine-like amino acid biosynthesis
BBa_K814007     ScyA (acetolactate synthase) generator
BBa_K814008     ScyB (leucine dehydrogenase) generator
BBa_K814009     ScyC generator
BBa_K814010     partial scytonemin biosynthetic pathway, scyCB
BBa_K814011     scytonemin biosynthetic pathway, scyBAC

Results

Figure 2. HPLC analysis of E. coli expressing MAA biosynthetic genes. Cell extracts of E. coli transformed with +DOA (DHQS, O-methyl transferase, and ATP-grasp) and +DOAN (DOA, and NRPS) showed production and 4-deoxygadusol (a) and mycosporine-glycine (b). Shinorine was not detected in the +DOAN expressing cells. Samples were compared against a +DA negative control (this construct lacks the ability to produce the 4-deoxygadusol intermediate). Cells expressing +ABC (ScyA, ScyB, ScyC) did not show scytonemin production. Data was compared with previous results from Balskus and Walsh, as a standard could not be obtained.

Figure 3. UV sensitivity screens of E. coli cultures producing MAAs. Preliminary results for the secondary screen showed increased survival in cells producing mycosporine-glycine when compared with +DO and +DA negative controls. Cells expressing +ABC did not show increased survivability.

Figure 4. Visual inspection of E. coli cells containing the scytonemin pathway. Although HPLC and growth tests were negative, cells expressing +ABC were seen to be expressing a deep brown product that could easily be visualized (below). The control is a cell culture expressing a +DA plasmid vector.

Conclusion
Successful transformation and expression of the mycosporine-glycine/shinorine pathway was achieved, and production of mycosporine-glycine was observed via HPLC analysis. This appears to incur resistance to UV-radiation to a certain degree. As we move forward, it will become critical to obtain expression of NRPS, the final gene in this pathway, and production of shinorine. Steps are being taken to optimize of this final step in the biosynthetic pathway is ongoing.

Although conclusive evidence regarding the production of scytonemin was not found, the initial results are promising. Previous studies on algae described a deep brown secretion in cells that were shown to be producing this compound. This pathway has not yet been fully characterized, and additional genes with unknown functions are present within the native N. punctiforme gene cluster. We are currently looking into cloning and expressing these additional genes, and looking for ways to isolate and analyze potential intermediate products within this pathway.


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