Team:MIT

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Latest revision as of 09:46, 30 October 2008

Home	The Team	The Project	Experiments	Parts Submitted to the Registry	Results	Notebook

Biogurt: A Sustainable and Savory Drug Delivery System

Streptococcus mutans, the main cause of dental caries, binds to glycoproteins on the teeth. A clinical study (Kelly CG et al.; Nature Biotechnol. 1999) isolated the 20aa functional segment (p1025) that S.mutans uses to attach to the teeth. p1025 competitively inhibits the binding of S.mutans, causing unharmful bacteria to grow in its place, preventing the recolonization of S.mutans for 90 days.

We are engineering Lactobacillus bulgaricus, a bacteria commonly found in yogurt, to produce and secrete this peptide under a promoter activated by lactose.

The peptide p1025 could simply be added to any food. But production of this peptide by L. bulgaricus is an independent process, so inserting the gene into live bacteria in yogurt will enable continuous production. Since a new batch of yogurt can be made using the bacteria from a small amount of the old batch, a continuous supply of teeth-cleaning yogurt will be available from the first successfully engineered batch. This could be the key to providing effective dental health care in underdeveloped rural communities, especially if yogurt is already an integral part of the diet.

Also, the p1025 gene could be replaced by any other gene, so this same expression system could be used to produce other useful peptides. Yogurt with modified bacteria will provide a cheap, efficient, and delicious way to distribute vitamins, vaccines and more.

Results

Characterization

plasmid pTG262 - Last year's Edinburgh team worked with the plasmid pTG262 in trying to transform it into different gram-positive bacteria. They had not transformed it into Lactobacillus but were optimistic that it could be, since pTG262 is known to be able to replicate in strains of Lactobacillus. Dr. Chris French was very helpful and graciously supplied us with this plasmid. Working extensively with this plasmid with all of our strains of bacteria, we were unable to successfully transform pTG262 into Lactobacillus, and we have concluded that it is our opinion that pTG262 cannot be electroporated into Lactobacillus delbruckii. We have added characterization to this part's main page and user review page, [http://partsregistry.org/wiki/index.php?title=Part:BBa_I742103 here].

p1025-This sequence codes for a short peptide, found to competitively inhibit binding of S.mutans to the tooth surface (CG et al.; Nature Biotechnol. 1999). S.mutans takes in sugars and secretes lactic acid, causing dental cavities, so introduction of this peptide into the mouth prevents colonies. This part uses the modified Silver BioBrick prefix and suffix to allow for protein construction.

Models

Competitive Binding Model

This model shows that p1025 inhibits binding of s. mutans to the salivary receptors on the tooth surface. p1025 serves as the competitive peptide inhibitor of bacterial adhesion in this model. Our team is the first to not only invent a tooth binding assay but characterize p1025 as a competitive inhibitor.

New Methods

S.mutans Binding Assay
p1025 Competitive Binding Assay
Novel Tooth Binding Assay
Lactococcus lactis Transformation Protocol
[http://openwetware.org/wiki/L._acidophilus_transformation Lactobacillus acidophilus Transformation Protocol]
[http://openwetware.org/wiki/Lactobacillus_transformation Lactobacillus delbruckii Transformation Protocol]
[http://openwetware.org/wiki/Lactobacillus_culture Culturing Lactobacillus]
[http://openwetware.org/wiki/Lactobacillus_miniprep Lactobacillus delbruckii Miniprep protocol]
[http://openwetware.org/wiki/Lactobacillus_planarum_miniprep Lactobacillus plantarum Miniprep protocol]

Lactobacillus Work

We successfully transformed Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis with a modified version of Serror et al's electrotransformation procedure. Our [http://openwetware.org/wiki/Lactobacillus_transformation transformation protocols] are found here. We also plan to submit these plasmids, with biobrick sites inserted in them, to the registry. These plasmids are [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128008 pJK650] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128007 pLEM415].

We are currently working on a method to effectively miniprep plasmid DNA from our Lactobacillus. For information about our protocol for doing so, please click [http://openwetware.org/wiki/Lactobacillus_miniprep here].

We have built an expression system for Lactobacillus to secrete protein, including individual biobricked parts of a Lactobacillus [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128006 promoter] and a Lactobacillus [http://partsregistry.org/Part:BBa_K128004 signal sequence].

Please visit the results page and experiments page of this wiki for full report of all our results and team-generated protocols!!!!

Acknowledgements

Graduate Advisers

Scott Carlson, Felix Moser, Chia-Yung Wu, Lav Varshney, Vikramaditya Yadav, Woo Chung, Rachel Hilmer, Robbier Barbero, Brian Cook, Jyoti Goda, Laure-Anne Ventouras

Faculty Advisors:

Drew Endy, Tom Knight

Others

Isadora Deese, Dr. Pascale Serror at L'Institut National de la Recherche Agronomique, Dr. Bernhard Heinrich at the University of Kaiserslautern, Dr. Chris French at the University of Edinburgh, Dr. Daniel Smith at the Forsyth Institute, Joey Davis at the Sauer Lab at MIT, The Fink Lab at the Whitehead Institute, Emiko Bare at the Keating Lab at MIT, Jennifer Hou at the Baker Lab at MIT, The UROP office and Biological Engineering Department for financial support

Home	The Team	The Project	Experiments	Parts Submitted to the Registry	Modeling	Notebook

@@ Line 11: / Line 11: @@
 !align="center"|[[Team:MIT/Team|The Team]]
 !align="center"|[[Team:MIT/Project|The Project]]
+!align="center"|[[Team:MIT/Experiments|Experiments]]
 !align="center"|[[Team:MIT/Parts|Parts Submitted to the Registry]]
-!align="center"|[[Team:MIT/Modeling|Modeling]]
+!align="center"|[[Team:MIT/Modeling|Results]]
 !align="center"|[[Team:MIT/Notebook|Notebook]]
 |}
@@ Line 19: / Line 20: @@
 <hr class=divider>
 <div id="about">
-{|
-| rowspan=2 |
-|[[Image:Teambiogurt.jpg|200px|right|frame]]
+==Biogurt: A Sustainable and Savory Drug Delivery System==
-|
+'''
-'''Welcome to the MIT team Wiki for iGEM 2008'''
+Streptococcus mutans, the main cause of dental caries, binds to glycoproteins on the teeth. A clinical study (Kelly CG et al.; Nature Biotechnol. 1999) isolated the 20aa functional segment (p1025) that S.mutans uses to attach to the teeth. p1025 competitively inhibits the binding of S.mutans, causing unharmful bacteria to grow in its place, preventing the recolonization of S.mutans for 90 days.
-*'''iGEM''' is the international genetically engineered machines competition.
-*The objective of the competition is to design and build an engineered biological system using [http://en.wikipedia.org/wiki/DNA DNA].
-*To see examples of the '''amazing possibilities''' of iGEM, check out last years [http://parts.mit.edu/igem07/index.php/Main_Page iGEM page]
-*Read our '''promotional brochure''' to learn about synthetic biology at MIT ([[media:mit_igem2008_brochure_front.gif|front]] and [[media:mit_igem2008_brochure_back.gif|back]]).
-*iGEM at MIT is possible because of '''outside support''', contact Tom Knight (tk [at] mit.edu) to help out!
-*iGEM at MIT is partly supported by the [http://web.mit.edu/urop/basicinfo/ Undergraduate Research Opportunity Program] and faculty including Drew Endy and Tom Knight.
-*We guarantee UROP funding for undergrads.  We need help to continue supporting undergrads, paying registration fees, and supplying lab reagents
-*'''Read more''' about our team on our '''[[/Notebook/Fundraise|fundraising notebook]]'''
-*<html>
+We are engineering Lactobacillus bulgaricus, a bacteria commonly found in yogurt, to produce and secrete this peptide under a promoter activated by lactose.
-<script language="JAVASCRIPT"><!-- update your browser, silly-->
-Today = new Date();
+The peptide p1025 could simply be added to any food. But production of this peptide by L. bulgaricus is an independent process, so inserting the gene into live bacteria in yogurt will enable continuous production. Since a new batch of yogurt can be made using the bacteria from a small  amount of the old batch, a continuous supply of teeth-cleaning yogurt will be available from the first successfully engineered batch. This could be the key to providing effective dental health care in underdeveloped rural communities, especially if yogurt is already an integral part of the diet.
-Jamboree = new Date("November 8, 2008");
-msInADay = 1000 * 60 * 60 * 24;
+Also, the p1025 gene could be replaced by any other gene, so this same expression system could be used to produce other useful peptides.  Yogurt with modified bacteria will provide a cheap, efficient, and delicious way to distribute vitamins, vaccines and more.
-display = Math.floor((Jamboree.getTime() - Today.getTime())/msInADay);
+'''
-document.write("There are " + display +" days left until the Jamboree!");
-// </script>
+==Results==
-</html>
+====Characterization====
-|}
+*plasmid pTG262 - Last year's Edinburgh team worked with the plasmid pTG262 in trying to transform it into different gram-positive bacteria. They had not transformed it into ''Lactobacillus'' but were optimistic that it could be, since pTG262 is known to be able to replicate in strains of ''Lactobacillus''. Dr. Chris French was very helpful and graciously supplied us with this plasmid. Working extensively with this plasmid with all of our strains of bacteria, we were unable to successfully transform pTG262 into ''Lactobacillus'', and we have concluded that it is our opinion that pTG262 '''cannot''' be electroporated into ''Lactobacillus delbruckii''. We have added '''characterization''' to this part's main page and user review page, [http://partsregistry.org/wiki/index.php?title=Part:BBa_I742103 '''here'''].
-</div>
-<hr class=divider>
+*  p1025-This sequence codes for a short peptide, found to competitively inhibit binding of S.mutans to the tooth surface (CG et al.; Nature Biotechnol. 1999). S.mutans takes in sugars and secretes lactic acid, causing dental cavities, so introduction of this peptide into the mouth prevents colonies. This part uses the modified Silver BioBrick prefix and suffix to allow for protein construction.
-==Project Description==
-''' Our goal is to engineer yogurt to prevent cavities.
+====Models====
+[[Team:MIT/Competitive Binding Model|Competitive Binding Model]]
+[[Image:Competitve binding.jpg]]
+*This model shows that p1025 inhibits binding of s. mutans to the salivary receptors on the tooth surface. p1025 serves as the competitive peptide inhibitor of bacterial adhesion in this model. Our team is the first to not only invent a tooth binding assay but characterize p1025 as a competitive inhibitor.
+====New Methods====
+*''S.mutans'' Binding Assay
+*p1025 Competitive Binding Assay
+* [[Team:MIT/Tooth_binding_assay_protocol|Novel Tooth Binding Assay]]
+* [[Team:MIT/Transforming_Lactococcus lactis|''Lactococcus lactis'' Transformation Protocol]]
+* [http://openwetware.org/wiki/L._acidophilus_transformation ''Lactobacillus acidophilus'' Transformation Protocol]
+* [http://openwetware.org/wiki/Lactobacillus_transformation ''Lactobacillus delbruckii'' Transformation Protocol]
+* [http://openwetware.org/wiki/Lactobacillus_culture Culturing ''Lactobacillus'']
+* [http://openwetware.org/wiki/Lactobacillus_miniprep ''Lactobacillus delbruckii'' Miniprep protocol'']
+* [http://openwetware.org/wiki/Lactobacillus_planarum_miniprep ''Lactobacillus plantarum'' Miniprep protocol'']
+====Lactobacillus Work====
+*We '''successfully transformed Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis''' with a modified version of Serror et al's electrotransformation procedure.  Our [http://openwetware.org/wiki/Lactobacillus_transformation '''transformation protocols'''] are found here. We also plan to submit these plasmids, with biobrick sites inserted in them, to the registry. These plasmids are [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128008 '''pJK650'''] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128007 '''pLEM415'''].
+*We are currently working on a method to effectively miniprep plasmid DNA from our ''Lactobacillus''. For information about our protocol for doing so, please click [http://openwetware.org/wiki/Lactobacillus_miniprep '''here'''].
+*We have built an expression system for Lactobacillus to secrete protein, including individual biobricked parts of a Lactobacillus [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128006 '''promoter'''] and a Lactobacillus [http://partsregistry.org/Part:BBa_K128004 '''signal sequence'''].
+*Please visit the results page and experiments page of this wiki for full report of all our results and team-generated protocols!!!!
+==Acknowledgements==
+=====Graduate Advisers=====
+Scott Carlson, Felix Moser, Chia-Yung Wu, Lav Varshney, Vikramaditya Yadav, Woo Chung, Rachel Hilmer, Robbier Barbero, Brian Cook, Jyoti Goda, Laure-Anne Ventouras
+=====Faculty Advisors:=====
+Drew Endy, Tom Knight
-Teeth are covered in a complex biofilm containing many types of bacteria and glycoproteins. Of all the bacteria found in the biofilm, only one strain, ''Streptococcus mutans'' is proven to be harmful. ''S.mutans'' attatches to a glycoprotein in the biofilm, forms colonies, takes in sugars, and secretes lactic acid which breaks down the enamel of the teeth, causing dental cavities.  A clinical study (Kelly CG et al.; Nature Biotechnol. 1999) isolated the protein that ''S.mutans'' uses to attach to the teeth and isolated the 20aa functional segment (p1025) that actually binds to the glycoproteins. p1025 competitively inhibits the binding of ''S.mutans'', causing other (unharmful) bacteria to grow in its place. This was found to prevent the recolonization of ''S.mutans'' for about 90 days.
+=====Others=====
-We are engineering ''Lactobacillus bulgaricus'', a bacteria commonly found in yogurt, to produce this peptide. Although the peptide could be commercially added to the yogurt, since production of this peptide by ''L. bulgaricus'' is independent, this mechanism will enable continuous production and remove the need for continuous addition of the peptide. Since a new batch of yogurt is made by using the bacteria from a small saved amount of the old batch, a continuous supply of teeth-cleaning yogurt will be made available immediately once the first batch is successfully engineered. The implications for such a biological system are enormous as it could revolutionalize dental health care around the world. This creation is applicable to and useful for all people; most importantly this could be the key to surpassing concerns of cost and availability of necessary and effective dental health care in small-scale, underdeveloped rural communities. '''
+Isadora Deese,
+Dr. Pascale Serror at L'Institut National de la Recherche Agronomique,
+Dr. Bernhard Heinrich at the University of Kaiserslautern,
+Dr. Chris French at the University of Edinburgh,
+Dr. Daniel Smith at the Forsyth Institute,
+Joey Davis at the Sauer Lab at MIT,
+The Fink Lab at the Whitehead Institute,
+Emiko Bare at the Keating Lab at MIT,
+Jennifer Hou at the Baker Lab at MIT,
+The UROP office and Biological Engineering Department for financial support
 |-
@@ Line 66: / Line 96: @@
 !align="center"|[[Team:MIT/Team|The Team]]
 !align="center"|[[Team:MIT/Project|The Project]]
+!align="center"|[[Team:MIT/Experiments|Experiments]]
 !align="center"|[[Team:MIT/Parts|Parts Submitted to the Registry]]
 !align="center"|[[Team:MIT/Modeling|Modeling]]
 !align="center"|[[Team:MIT/Notebook|Notebook]]
 |}