CHE.496/2009/Schedule/Oral presentations of part design/Group 1

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** The basic idea is that we would use the unique properties that thermophile organisms have with stable DNA/RNA/Proteins at high temperatures that would normally denature bacterial organisms and build a vector with those properties. The idea is that if we are able to produce a protein that is very stable at high temperatures in an organism that is not a thermophile such as E. Coli; we then could denature the cells through heat and all the proteins would break down into secondary structures and so on. It would be a simple matter of separating the denatured proteins from the target protein that is not denatured through SDS-Page gel (based on molecular weight) or some other method. If there was a desirable protein that was difficult to purify by conventional methods such as ion exchange columns, this method could be used.  Thermostable proteins have higher charged amino acid content than average and proteins have more salt bridges in them.  The proteins also use hydrophobic interactions to stabilize. The manner we use to confer the targeted enzyme sequence for our desired protein with the properties of stability at high temperatures will have to be studied further.  This project could also have have the upside of enabling the cheap growth of thermostable proteins which are generally expensive because of the difficulty in growing extremophiles in the laboratory.
** The basic idea is that we would use the unique properties that thermophile organisms have with stable DNA/RNA/Proteins at high temperatures that would normally denature bacterial organisms and build a vector with those properties. The idea is that if we are able to produce a protein that is very stable at high temperatures in an organism that is not a thermophile such as E. Coli; we then could denature the cells through heat and all the proteins would break down into secondary structures and so on. It would be a simple matter of separating the denatured proteins from the target protein that is not denatured through SDS-Page gel (based on molecular weight) or some other method. If there was a desirable protein that was difficult to purify by conventional methods such as ion exchange columns, this method could be used.  Thermostable proteins have higher charged amino acid content than average and proteins have more salt bridges in them.  The proteins also use hydrophobic interactions to stabilize. The manner we use to confer the targeted enzyme sequence for our desired protein with the properties of stability at high temperatures will have to be studied further.  This project could also have have the upside of enabling the cheap growth of thermostable proteins which are generally expensive because of the difficulty in growing extremophiles in the laboratory.
*Source:
*Source:
-
[http://www.asm.org/ASM/files/CCLIBRARYFILES/FILENAME/0000000297/6403p137.pdf Microbiology in Yellowstone]
+
**[http://www.asm.org/ASM/files/CCLIBRARYFILES/FILENAME/0000000297/6403p137.pdf Microbiology in Yellowstone]
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[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1574309&blobtype=pdf Analysis of Nanoarchaeum equitans genome and proteome composition: indications for hyperthermophilic and parasitic adaptation]
+
**[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1574309&blobtype=pdf Analysis of Nanoarchaeum equitans genome and proteome composition: indications for hyperthermophilic and parasitic adaptation]
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[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4JT3FRX-2&_user=709071&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000039638&_version=1&_urlVersion=0&_userid=709071&md5=70ffc9f3a8143ec08f4a8075c771096d  Prediction of potential thermostable proteins in Xylella fastidiosa]
+
**[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4JT3FRX-2&_user=709071&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000039638&_version=1&_urlVersion=0&_userid=709071&md5=70ffc9f3a8143ec08f4a8075c771096d  Prediction of potential thermostable proteins in Xylella fastidiosa]
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**A vector would be built that consists of an operon that expresses this gene to produce large quantities of the protein glucocerebrosidase. The protein would have to be tagged in order to facilitate transport outside of the cell.  Insertion into the secretory pathway may be achieved by adding an amino terminal signal peptide.  The signal will direct the protein to the outside of the cellular membrane and then will be cleaved by a secreted peptidase.  Hopefully we would be able to use an endogenous peptidase.  The signal is composed of a short hydrophillic region followed by one or more positively charged residues and ending in a long hydrophobic tail. The chassis used to produce the protein would have to have a high copy number in order to produce as much protein as possible. The steps for protein purification from the growth media would have to be determined and to ensure that the protein is not bound to the surface of the lipid bilayer cell membrane.   
**A vector would be built that consists of an operon that expresses this gene to produce large quantities of the protein glucocerebrosidase. The protein would have to be tagged in order to facilitate transport outside of the cell.  Insertion into the secretory pathway may be achieved by adding an amino terminal signal peptide.  The signal will direct the protein to the outside of the cellular membrane and then will be cleaved by a secreted peptidase.  Hopefully we would be able to use an endogenous peptidase.  The signal is composed of a short hydrophillic region followed by one or more positively charged residues and ending in a long hydrophobic tail. The chassis used to produce the protein would have to have a high copy number in order to produce as much protein as possible. The steps for protein purification from the growth media would have to be determined and to ensure that the protein is not bound to the surface of the lipid bilayer cell membrane.   
*Sources:
*Sources:
-
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=2629 GBA glucosidase, beta]
+
**[http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=2629 GBA glucosidase, beta]
-
[http://www.ncbi.nlm.nih.gov/nuccore/223717990?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Nucleotide sequence]
+
**[http://www.ncbi.nlm.nih.gov/nuccore/223717990?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Nucleotide sequence]
-
[http://www.rcsb.org/pdb/explore/biologyAndChemistry.do?structureId=1OGS PDB]
+
**[http://www.rcsb.org/pdb/explore/biologyAndChemistry.do?structureId=1OGS PDB]
-
[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=207531&blobtype=pdf  Stabilization of Phosphorylated Bacillus subtilis DegU by DegR]
+
**[http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=207531&blobtype=pdf  Stabilization of Phosphorylated Bacillus subtilis DegU by DegR]
-
[http://mmbr.asm.org/cgi/reprint/57/1/50  The Complete General Secretory Pathway in
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**[http://mmbr.asm.org/cgi/reprint/57/1/50  The Complete General Secretory Pathway in Gram-Negative Bacteria]
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Gram-Negative Bacteria]
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Current revision

CHE.496: Biological Systems Design Seminar

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Group 1

  • Members: Patrick Gildea & Thaddeus Webb

Parts

=Part 1=: Multiple input AND gate
  • Description:
    • The genetic circuit would function as a standard AND gate with the exception that it does not function on binary input but ternary and possibly quaternary input if we can make it possible. While it is possible to build a circuit that can encode multiple inputs by linking a series of AND gates in a cascade as has been done in electronic circuits and VLSI design. However, in a biological system there are stoichastic effects that can interfere with a large biological network. Another consideration is that for such a network of multiple binary AND gates in a biological organism, the inputs would have to be different from each other because otherwise it would defeat the purpose of the genetic circuits. Having an AND gate with multiple inputs would eliminate the issues listed above and would be far more efficient and faster. The AND gate could be constructed of genes or possibly orthogonal Ribosomes.
  • Source & References


=Part 2=: Repressilator (different design)
  • Description:
    • The genetic circuit would function similarly to a repressilator as designed by Elowitz and Leibler but it would be of a different design to accomplish the same purpose. Instead of using three transcriptional repressor systems to repress each other in a cycle, the new design would consist of two oscillating circuits that would cycle between three states, multiple states would be possible. In the construction of the oscillating circuits, one circuit can be designed to be dependent on an input substrate such as the lactose or arabinose promoter. The advantage this new design would confer more stability between transitions from one state to another and would be more uniform in system response (hopefully). In other words, fluctuations between states would be minimized where one state would not be active for a longer time than another state. Such a system could be insulated in the cell and used for biological network analysis for systems biology.
  • Source:

A synthetic oscillatory network of transcriptional regulators


=Part 3=:AND-OR gate
  • Description:
    • This would be a three input AND gate which would require the presence of one inducer(1) and the presence of one of two other inducers(2 and 3). The gate could have two different outputs based on whether inducers 2 or 3 are present. An example of how this might be constructed would to have inducer1 activate for an orthagonal ribosome. The other two inducers can activate for any output function but the resulting mRNA could only be translated by the ribosome from inducer1. Therefore the outputs will only be active when inducer 1, 2, and 3 are present and what the output will be will depend on whether inducer 2 or 3 is present. This could be useful when tied with other logic gates because it would enable the cell to tie new information into the outputs of other gates. For example, if a cell were programmed to respond to an extracellular signal but different responses are required in acidic conditions or high salt conditions this gate could be linked to sensors and be used to decide whether to respond and if so, how.
  • Source:


=Part 4=: Protein-Purification by way of Thermophile polymerase
  • Description:
    • The basic idea is that we would use the unique properties that thermophile organisms have with stable DNA/RNA/Proteins at high temperatures that would normally denature bacterial organisms and build a vector with those properties. The idea is that if we are able to produce a protein that is very stable at high temperatures in an organism that is not a thermophile such as E. Coli; we then could denature the cells through heat and all the proteins would break down into secondary structures and so on. It would be a simple matter of separating the denatured proteins from the target protein that is not denatured through SDS-Page gel (based on molecular weight) or some other method. If there was a desirable protein that was difficult to purify by conventional methods such as ion exchange columns, this method could be used. Thermostable proteins have higher charged amino acid content than average and proteins have more salt bridges in them. The proteins also use hydrophobic interactions to stabilize. The manner we use to confer the targeted enzyme sequence for our desired protein with the properties of stability at high temperatures will have to be studied further. This project could also have have the upside of enabling the cheap growth of thermostable proteins which are generally expensive because of the difficulty in growing extremophiles in the laboratory.
  • Source:


=Part 5=: Production of GBA glucosidase in E. Coli
  • Purpose:
    • To produce beta-glucosidase, a lysosomal membrane protein that cleaves the beta-glucosidic linkage of glycosylceramide, an intermediate in glycolipid metabolism. Mutations in this gene cause Gaucher disease, a lysosomal storage disease characterized by an accumulation of glucocerebrosides.
  • Description of how the part would work:
    • A vector would be built that consists of an operon that expresses this gene to produce large quantities of the protein glucocerebrosidase. The protein would have to be tagged in order to facilitate transport outside of the cell. Insertion into the secretory pathway may be achieved by adding an amino terminal signal peptide. The signal will direct the protein to the outside of the cellular membrane and then will be cleaved by a secreted peptidase. Hopefully we would be able to use an endogenous peptidase. The signal is composed of a short hydrophillic region followed by one or more positively charged residues and ending in a long hydrophobic tail. The chassis used to produce the protein would have to have a high copy number in order to produce as much protein as possible. The steps for protein purification from the growth media would have to be determined and to ensure that the protein is not bound to the surface of the lipid bilayer cell membrane.
  • Sources:


=Part 6=: Iron absorption by E. Coli 
  • Purpose:
    • To enable E. Coli to absorb iron from the environment and survive. This could be useful both in the extraction of iron and in cleanup of a contaminated area.
  • Description of how the part would work:
    • This part would be based primarily on two genes VIT1 and ferretin. VIT1 is a transporter protein used by arabidopsis to uptake iron from the soil. Over expression of this single gene in arabidopsis has been shown to increase iron uptake by several hundred percent. Proper expression of this gene would require correct localization in the cell, specifically we would need to localize expression to the cell membrane. Plants and animals both use the protein ferritin to safely store iron in the cytoplasm. If ferritin could be properly expressed in bacteria it should allow them to survive with high intracellular concentrations of iron. This part would probably need to by used in conjunction with a motility part or another mechanism for collecting the cells.
  • Sources:
    • Description of iron transport[1]
    • Storage of iron by ferritin[2]
    • Iron transport and signalling in plants[3]



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