IGEM:Harvard/2006/Fusion proteins

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=Project Overview=
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*The cell surface streptavidin project approaches targeting from a different direction, that is, from the cell. While the adaptamer project uses nucleic acids to target any two substrates to each other, the cell surface streptavidin project seeks to target any substrate(s) to the cell surface.
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*The method of targeting would be implemented through the '''expression of streptavidin protein on the cell surface'''. Streptavidin is a protein which binds strongly to the biotin molecule, thus there is a common practice of biotinylating (adding biotin to) nucleic acids or peptides for streptavidin affinity purification or antibody conjugation. We would be utilizing this strong streptavidin-biotin binding to target <u>any</u> biotinylated nucleic acid or peptide to the outside of a cell expressing streptavidin on the surface. Here are some potentially useful applications.
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**bringing a biotinylated protein to the cell surface for facilitated interaction with some other surface element.
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**linking a cell with another protein/cell via a biotinylated aptamer.
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**delivering a DNA nanobox to the cell surface through a biotinylated oligonucleotide.
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__TOC__
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*Streptavidin would be expressed on the cell surface through the '''Lpp-OmpA surface display vehicle''' (Earhart, 2000). The complete vehicle consists of three fused protein domains.
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**Signal peptide of lipoprotein (Lpp). This targets the protein to the outer membrane.
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** Transmembrane domains of outer membrane protein A (OmpA). This spans the protein across the outer membrane to the cell surface.
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**The surface protein.
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=Plan=
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*Assembly of the fusion protein would be carried out through a '''modified BioBricks assembly for protein domains''' (Phillips and Silver, 2006). Standard BioBricks parts separated from the flanking XbaI and SpeI sites by a single spacer nucleotide, in order to prevent Dam methylation. If you leave out these spacer nucleotides, the mixed site formed between assembled parts is six base pairs long, and so reading frame can be maintained between assembled protein domains.
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See the slides for some brief info.
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==Express monomeric streptavidin on E. coli cell surface==
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'''Scaffold 1: GPI anchor'''
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<gallery>
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Image:Lpp ompa.JPG|Lpp-OmpA surface display vehicle (Francisco et al., 1993)
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Image:Protein domain biobrick.JPG|Modified BioBricks assembly for protein domains
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</gallery>
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'''Scaffold 2: Beta autotransporter'''
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=Results=
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*We completed assembly of constructs using the following BioBrick parts.
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**J04500. a composite part of a lac promoter (R0010) and a strong ribosome binding site (B0031)
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**J36835. Lpp, the lipoprotein signal peptide.
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**J36837 or J36838. OmpA, one (O1) or five (O5) transmembrane domains, respectively. Both have been shown to work (Earhart, 2000).
 +
**J36841 or J36843. Streptavidin, either wild-type "SW" (Howarth, 2006), or single-chain dimeric "SD" (Aslan, 2005).
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***Note: Streptavidin exists naturally as a soluble tetramer, but restriction to the surface might allow only monomeric/dimeric forms. What is interesting is that there has been research done to engineer such forms, in order to render biotin binding less strong but more easily reversible (Wu, 2005).
 +
*Western blots were performed with transformed and induced cells, probing with anti-his6 antibody (each streptavidin had a His6 tag) and with anti-streptavidin antibody. Distinct bands were observed at the expected sizes in anti-his6 probing, and bands were observed at the same places with the anti-streptavidin probing. These results suggest the following.
 +
**The promoter and ribosome binding site are functioning correctly for expression of these fusion protein construct.
 +
**The BioBricks assembly of the fusion protein was successful in that reading frame was maintained, since the coding sequence for the His6 tag is found at the end of the construct.
 +
**The streptavidin part of the fusion protein is still folding in such a way that anti-streptavidin antibody can recognize it.
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'''Scaffold 3: Lpp-OmpA'''
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<gallery>
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Image:Cell surface streptavidin construct.JPG|Diagram of cell surface streptavidin construct
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Image:Cell surface streptavidin westerns.JPG|Western blots of cell surface streptavidin constructs
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</gallery>
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<u>Major questions/concerns</u>
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=Future Plans=
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#Where can we get the DNA expressing the scaffold protein. I need to look more into this, but I don't think we can just order it.
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*Now that we know that the construct is being expressed, we need to determine whether or not the construct is being localized to the outer membrane. We can separate the cell lysate by centrifugation, isolate the outer membrane proteins, and then perform Western blots, probing with anti-his6 and anti-streptavidin antibody.
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#Where do we get streptavidin DNA (smaller problem)
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*If the construct is indeed being localized to the outer membrane, we then need to determine if the streptavidin is being displayed functionally on the cell surface. We can probe whole cells with anti-streptavidin antibodies or even fluorescently tagged streptavidin aptamers for in-cell westerns, or we can visualize surface binding of biotinylated, fluorescently tagged oligonucleotides under a microscope.
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#What constraints do we have to worry about with streptavidin? In the original and later autotransporter papers, the passenger protein was mutated to lack cysteines in order to prevent disulfide bond formation in the periplasm: tertiary structure prevents passage of the protein through the beta barrel.  
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*If streptavidin is being expressed on the cell surface, we can switch in other engineered streptavidin clones and compare biotin binding. We can also try adding a length of amino acids between OmpA and streptavidin, which might give spatial flexibility to allow formation of tetramers on the cell surface or to allow the streptavidin to extend outside of any extracellular complexes.
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#How much time to get a first experiment up and running? This depends on how much work we have to do with the scaffold protein sequence and how fast we can get them (likely a long time).
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#Will we be able to correspond with experts on this system? The founder of autodisplay, Joachim Jose, has not responded to some intial questions about autotransporter.
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==Demonstrate control of a nucleic acid interface between proteins and the cell surface==
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'''Notes'''
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(Maybe Un-, but more likely..) fortunately, another group has done work like this. We'll expand on the "adaptamer" concept of Tahiri-Alaoui et al. (12000850). In this paper, the group evolved one aptamer which bound streptavidin, then spliced it to a CopA RNA. Then they spliced a complementary CopT RNA to a CD4 aptamer. Following CopA/CopT binding, the resulting construct could bind both CD4 and streptavidin. Neato. CopA and CopT bind each other by a kissing complex between two loops; mutations have been introduced before, so looking at those studies might be a good starting places for adjusting the dissociation rate (16199086).
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The reason they go through the trouble of using CopA and CopT is that they maintain stable secondary structures; the group reports that when you just have any old complementary strands, the binding affinities of the aptamers goes down considerably. We may be able to design our own RNA-RNA interfaces to have well-defined secondary structure, enhancing our ability to control dissociation rate.
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Additionally, we want to make one of these aptamers bind to the E. coli cell surface. There is a serious dearth of known aptamers that do this. The only report about one is 15541352; their aptamer binds LPS, a major component of the E. coli outer membrane, so it has a lot of targets. They don't give a Kd value, maybe because it didn't matter too much with so much LPS. Hope we can get our hands on the sequence..
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One thing to consider: in the absence of aptamers that bind to the cell surface, we might as well try it with aptamers that bind proteins in vitro as done in 12000850. Why worry about cells for now? We're interested in a proof of principle.
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'''Initial experiment'''
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We'll attempt to build an adaptamer that can bind thrombin and streptavidin simultaneously. More details to come. Oligos to be used [[/thrombstrepadapt/|here]]
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<u>Major Questions/concerns</u>
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#What aptamers exist that bind the E. coli cell surface??? (and have public sequences! The sequence of the aptamer that binds LPS is not reported in the paper.)
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#Is there any chance that we'll be able to evolve our own aptamer this summer? Online resources point toward 'no' but see last question.
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#What other secondary structures might work as interfaces? If we design our own, the online programs mfold and Vienna RNA may be useful. Otherwise, we'll have to go with known complementary structures. Do the faculty members have any suggestions for structures?
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#Time and reliability: Hopefully short! Aptamer sequences are relatively short, so we'll likely be able to order them instead of asking groups for them. Then we'd try tons of intermediate interfaces, which is just a matter of ordering more oligos.
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#Will people respond to email? I've emailed David Liu about evolving aptamers and Sulatha Dwarakanath (author of 15541352) about aptamers that target the E. coli cell surface to no response. It would be quite helpful to hear their thoughts.
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==Other projects==
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A couple of related projects that we haven't really researched..
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#Having E. coli export an aptamer outside the cell
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#Using aptamers to actually stimulate a response on the cell surface
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=Presentations=
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[[Media:Protein_domain_biobricks_presentation,_june_19_2006.ppt|Protein domain BioBricks presentation]]
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[[Media:Targeting.ppt|Cell surface targeting Week 1]]
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[[Media:Cell_Surface_Targeting_7-10.ppt|Cell surface targeting Week 4]]
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=Working Team Members=
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*[[User:Perry/Summer_2006_Harvard_iGEM_work |Perry Tsai]] ([[User_talk:Perry|talk]])
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*[[User:Lhahn|Lewis Hahn]] ([[User_talk:Lhahn|talk]])
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=Recent Changes=
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{{Special:Recentchanges/b=IGEM:Harvard/2006/Fusion_proteins&limit=25}}
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Current revision


Project Overview

  • The cell surface streptavidin project approaches targeting from a different direction, that is, from the cell. While the adaptamer project uses nucleic acids to target any two substrates to each other, the cell surface streptavidin project seeks to target any substrate(s) to the cell surface.
  • The method of targeting would be implemented through the expression of streptavidin protein on the cell surface. Streptavidin is a protein which binds strongly to the biotin molecule, thus there is a common practice of biotinylating (adding biotin to) nucleic acids or peptides for streptavidin affinity purification or antibody conjugation. We would be utilizing this strong streptavidin-biotin binding to target any biotinylated nucleic acid or peptide to the outside of a cell expressing streptavidin on the surface. Here are some potentially useful applications.
    • bringing a biotinylated protein to the cell surface for facilitated interaction with some other surface element.
    • linking a cell with another protein/cell via a biotinylated aptamer.
    • delivering a DNA nanobox to the cell surface through a biotinylated oligonucleotide.
  • Streptavidin would be expressed on the cell surface through the Lpp-OmpA surface display vehicle (Earhart, 2000). The complete vehicle consists of three fused protein domains.
    • Signal peptide of lipoprotein (Lpp). This targets the protein to the outer membrane.
    • Transmembrane domains of outer membrane protein A (OmpA). This spans the protein across the outer membrane to the cell surface.
    • The surface protein.
  • Assembly of the fusion protein would be carried out through a modified BioBricks assembly for protein domains (Phillips and Silver, 2006). Standard BioBricks parts separated from the flanking XbaI and SpeI sites by a single spacer nucleotide, in order to prevent Dam methylation. If you leave out these spacer nucleotides, the mixed site formed between assembled parts is six base pairs long, and so reading frame can be maintained between assembled protein domains.

Results

  • We completed assembly of constructs using the following BioBrick parts.
    • J04500. a composite part of a lac promoter (R0010) and a strong ribosome binding site (B0031)
    • J36835. Lpp, the lipoprotein signal peptide.
    • J36837 or J36838. OmpA, one (O1) or five (O5) transmembrane domains, respectively. Both have been shown to work (Earhart, 2000).
    • J36841 or J36843. Streptavidin, either wild-type "SW" (Howarth, 2006), or single-chain dimeric "SD" (Aslan, 2005).
      • Note: Streptavidin exists naturally as a soluble tetramer, but restriction to the surface might allow only monomeric/dimeric forms. What is interesting is that there has been research done to engineer such forms, in order to render biotin binding less strong but more easily reversible (Wu, 2005).
  • Western blots were performed with transformed and induced cells, probing with anti-his6 antibody (each streptavidin had a His6 tag) and with anti-streptavidin antibody. Distinct bands were observed at the expected sizes in anti-his6 probing, and bands were observed at the same places with the anti-streptavidin probing. These results suggest the following.
    • The promoter and ribosome binding site are functioning correctly for expression of these fusion protein construct.
    • The BioBricks assembly of the fusion protein was successful in that reading frame was maintained, since the coding sequence for the His6 tag is found at the end of the construct.
    • The streptavidin part of the fusion protein is still folding in such a way that anti-streptavidin antibody can recognize it.

Future Plans

  • Now that we know that the construct is being expressed, we need to determine whether or not the construct is being localized to the outer membrane. We can separate the cell lysate by centrifugation, isolate the outer membrane proteins, and then perform Western blots, probing with anti-his6 and anti-streptavidin antibody.
  • If the construct is indeed being localized to the outer membrane, we then need to determine if the streptavidin is being displayed functionally on the cell surface. We can probe whole cells with anti-streptavidin antibodies or even fluorescently tagged streptavidin aptamers for in-cell westerns, or we can visualize surface binding of biotinylated, fluorescently tagged oligonucleotides under a microscope.
  • If streptavidin is being expressed on the cell surface, we can switch in other engineered streptavidin clones and compare biotin binding. We can also try adding a length of amino acids between OmpA and streptavidin, which might give spatial flexibility to allow formation of tetramers on the cell surface or to allow the streptavidin to extend outside of any extracellular complexes.
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