IGEM:Harvard/2006/Fusion proteins

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[[Media:Protein_domain_biobricks_presentation,_june_19_2006.ppt|Protein domain BioBricks presentation]]
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<div class="tabs">
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<ul>
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<li id="current">[[IGEM:Harvard/2006/Fusion proteins|1. Main Page]]</li>
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<li>[[IGEM:Harvard/2006/Fusion proteins/Notebook|2. Lab Notebook]]</li>
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<li>[[IGEM:Harvard/2006/Fusion proteins/Literature|3. Literature]]</li>
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<li>[[IGEM:Harvard/2006/Fusion proteins/Oligos|4. Oligos]]</li>
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</ul>
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</div>
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<br style="clear:both">
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<div class="tabcontent">
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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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[[Media:Targeting.ppt|Cell surface targeting]]
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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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A suggestion: although it might be bad form, when citing an article, just refer to the PMID. This is more useful than the pretty pubmed link which isn't smart enough to know when you are logged in with a HUID.
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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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=Plan=
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<gallery>
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See the slides for some brief info.
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Image:Lpp ompa.JPG|Lpp-OmpA surface display vehicle (Francisco et al., 1993)
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==Express monomeric streptavidin on E. coli cell surface==
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Image:Protein domain biobrick.JPG|Modified BioBricks assembly for protein domains
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===Scaffold 1: GPI anchor===
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</gallery>
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We've looked at this less so far since autotransporter seemed more attractive, but maybe we should look at it more.
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===Scaffold 2: Beta autotransporter===
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See some of the readings below for more info.
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===Major questions/concerns===
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=Results=
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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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*We completed assembly of constructs using the following BioBrick parts.
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#Where do we get streptavidin DNA (smaller problem)
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**J04500. a composite part of a lac promoter (R0010) and a strong ribosome binding site (B0031)
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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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**J36835. Lpp, the lipoprotein signal peptide.
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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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**J36837 or J36838. OmpA, one (O1) or five (O5) transmembrane domains, respectively. Both have been shown to work (Earhart, 2000).
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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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**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).
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*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.
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**The promoter and ribosome binding site are functioning correctly for expression of these fusion protein construct.
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**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.
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**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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==Demonstrate control of a nucleic acid interface between proteins and the cell surface==
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<gallery>
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===Notes===
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Image:Cell surface streptavidin construct.JPG|Diagram of cell surface streptavidin construct
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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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Image:Cell surface streptavidin westerns.JPG|Western blots of cell surface streptavidin constructs
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</gallery>
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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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=Future Plans=
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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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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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*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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*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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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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===Major Questions/concerns===
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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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=Reading=
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==General Fusion==
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<biblio>
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#fusion1 pmid=1367360
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# fusion1ab We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.
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#fusion2 pmid=2476847
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#fusion3 pmid=2548185
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#fusion4 pmid=3079747
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#fusion5 pmid=7691170
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#fusion6 pmid=15695809
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#fusion6ab Bradyrhizobium japonicum is an important nitrogenfixing symbiotic bacterium, which can form root nodules on soybeans. These bacteria have a gene encoding a putative avidin- and streptavidin-like protein, which bears an amino acid sequence identity of only about 30% over the core regions with both of them. We produced this protein in Escherichia coli both as the full-length wild type and as a C-terminally truncated core form and showed that it is indeed a high affinity biotin-binding protein that resembles (strept)avidin structurally and functionally. Because of the considerable dissimilarity in the amino acid sequence, however, it is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin did not show significant cross-reactivity with it. Therefore this new avidin, named bradavidin, facilitates medical treatments such as targeted drug delivery, gene therapy, and imaging by offering an alternative tool for use if (strept)avidin cannot be used, because of a deleterious patient immune response for example. In addition to its medical value, bradavidin can be used both in other applications of avidin-biotin technology and as a source of new ideas when creating engineered (strept)avidin forms by changing or combining the desired parts, interface patterns, or specific residues within the avidin protein family. Moreover, the unexpected discovery of bradavidin indicates that the group of new and undiscovered bacterial avidin-like proteins may be both more diverse and more common than hitherto thought.
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</biblio>
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<br>
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==E. coli cell surface display==
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<biblio>
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#display1 pmid=16369779
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#display2 pmid=11024362
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#display3 pmid=9624691
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#display4 pmid=12039531
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#display5 pmid=
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</biblio>
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<br>
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===Notes on Autodisplay review (display1) 16369779===
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This is a review about the Autodisplay system used to express proteins on the E. coli cell surface. Some of the important features of this system are:
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*Utilizes E.coli-native AIDA-I as scaffold
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*Detection: monoclonal antibodies and protease cleavage sites created
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*Protein unfolded during transport to cell surface.
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*Variety of proteins have been displayed (p.610)
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*Possibly to display catalytically active enzymes.
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*Dimerization of proteins has been observed! (unique to this system); work on tetramers in progress. Protein anchor floats around membrane.
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*Many proteins displayed: ~10^5 without loss of cell viability
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The writer claims that the system is superior compared to all other display systems, but he might be biased since he created it.
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===Notes on expressing genes in different compartments review (display2) 11024362===
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This is a more general review which is actually mostly about cell-surface display techniques. It offers a wider range of options, but came out in 2000, so new options probably have become available.
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*5 options; a good summary is found on page 3.
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**Porins- Insert protein <= 60 residues
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**Fimbriae- Insert protein <= 15 residues
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**Lipoproteins
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**GPI anchor
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**Beta-autotransporter - same system as that used in display1
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*The latter three were claimed to support large polypeptides. However, looking at a few of the articles seemed to suggest that they actually only tried them out with small polypeptides. The winners appear to be GPI anchors and beta autotransporters, although this might have changed in recent years. See display3 for the use of GPI anchors.
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===GPI anchor(display3) 9624691===
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I didn't read this one that thoroughly, but the point is that they forced E. coli to express functional levansucrase bound to a GPI anchor, Inp. Using these cells, they successfully converted sucrose to levan, which seems neat. Levansucrase, is about 400 a.a. residues long.
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===More bacterial display (display4) 12039531===
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This review's gram-negative bacteria section is slightly more recent than display1 but contains almost no more information. It does have more explicit weight constraints on the proteins you can display for each system and pays slightly more attention to other constraints, but nothing that interesting. It has an equally comprehensive section on gram-positive bacteria. You might as well look at this one and not display1, though because it was published in 2002 vs. 2000 for display1.
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It does mention that gram-positive bacteria are more suitable for display. Tested gram-postive display systems are better at displaying large proteins. Additionally, protein only need pass through one membrane versus moving through the cytoplasmic membrane and achieving the correct orientation in the outer membrane. Finally, gram-positive bacteria are more durable and easier to test under harsh lab procedures.
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==Aptamers==
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<biblio>
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#apt1 pmid=15541352
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#apt2 pmid=11808691
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#apt3 pmid=12000850
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#apt4 pmid=16199086
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#apt5 pmid=10744017
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</biblio>
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<br>
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===Quantum dots conjugated to aptamers (apt1) 15541352===
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This was a neat study in which quantum dots were conjugated to antibodies or aptamers that target E. coli cell surfaces. The point of the article is that the emissions from the quantum dots changed following binding, but what's relevant to us is the aptamer they used. They evolved it on their own using the method of apt2 (probably out of convenience: both were Austin-based companies). The aptamer recognizes lipopolysaccharide (LPS O111:B4). I am currently emailing to find out why they chose LPS. At least we know of one aptamer that binds the E. coli cell surface...
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===Adaptamers (apt3) 12000850===
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This paper is also listed in the streptavidin section. See "Notes" under "Demonstrate Control.." project for a description.
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===CopA/CopT (apt4) 16199086===
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This recent review paper has a description of the CopA/CopT interaction which is utilized in apt3.
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===CopA/CopT (apt5) 10744017===
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This paper reveals the extremely intricate "deep-kissing" structure formed between CopA and CopT.
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==Streptavidin==
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<biblio>
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#sa1 pmid=2201874
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#sa2 pmid=2404273
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#sa3 pmid=2406253
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#sa4 pmid=2025272
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#sa5 pmid=7499314
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#sa6 pmid=9113646
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#sa7 pmid=9037005
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#sa8 pmid=9177186
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#sa9 pmid=10796996
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#sa10 pmid=11036649
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#sa11 pmid=11959132
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#sa12 pmid=10356256
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#sa13 pmid=11959132
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#sa14 pmid=15695809
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#sa15 pmid=12000850
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#sa16 pmid=16554826
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#sa17 pmid=15840576
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#sa18 pmid=16554831
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#sa19 pmid=12182820
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#sa20 pmid=11584006
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#sa21 pmid=11345441
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#sa22 pmid=15939877
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#sa23 pmid=16289701
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#sa24 pmid=9790881
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#sa25 pmid=15845380
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#sa26 pmid=14217155
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</biblio>
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<br>
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===Display of Tetrameric streptavidin on B. subtilis spore surfaces (sa23) 16289701===
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As the title sugggests, a group was actually able to express _tetrameric_ streptavidin on the B. subtilis cell surface. This was a lot easier than doing it in other types of bacteria since they didn't have to worry about getting across the cell membrane or cell wall: when B. subtilis forms spores, a bunch of proteins form in the cytosol which then become the coats of spores... Additionally, spores form approximately when cells stop dividing, which somehow defeats the biotin sequestration problem. Actually, they never do a specific experiment to show this, but we are probably supposed to infer that it was enough to just see the streptavidin on the surface(?) Regardless, this organism is pretty different from E. coli, so it probably isn't terribly important.
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==ompA==
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<biblio>
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#ompA1 pmid=16391137
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#ompA2 pmid=9005446
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#ompA3 pmid=8920186
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#ompA4 pmid=6313361
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</biblio>
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==Related articles==
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<biblio>
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#other1 pmid=9893944
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</biblio>
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<br>
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===Cd2+ binding (other1) 9893944===
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other1 is an old review article I came across talking about expressing metallothioneins on the cell surface of E. coli; these things bind Cd2+, so those interested in pollution cleanup might want to take a look.
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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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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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