IGEM:Harvard/2006/Fusion proteins

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I have contacted Filiz Aslan at DFCI, and she has agreed to give me clones of her single-chain dimeric streptavidin (Aslan et al., 2005). I'm in the process of having the MTA (Material Transfer Agreement) approved by Harvard's Office of Technology Development. Once the approval has gone through, we can pick up the clone!

I've also gotten a reply from Dr. Takeshi Sano, also at DFCI. He has pTSA13-wt, which is wild-type streptavidin clone. Another MTA will have to be approved.

I have word from Dr. Charles Earhart that his colleague Dr. George Georgiou "has been distributing LppOmpA vector plasmids and has a large collection." I've emailed Dr. Georgiou several times without reply. I tried emailing his administrative assistant Donna, and she replied she would pass it along to someone in the lab. Closer? Maybe.

We may be able to make Dr. Georgiou's construct ourselves. It is a fusion protein N - Lpp aa's 1-9 - OmpA aa's 46-159 - surface protein - C. E. coli K12 genome and BioBrick primers for the full OmpA gene (start through stop) have been ordered.

Perry 14:45, 30 June 2006 (EDT)



See the slides for some brief info.

Express monomeric streptavidin on E. coli cell surface

Scaffold 1: GPI anchor

Scaffold 2: Beta autotransporter

Scaffold 3: Lpp-OmpA

Major questions/concerns

  1. 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.
  2. Where do we get streptavidin DNA (smaller problem)
  3. 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.
  4. 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).
  5. 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.

Demonstrate control of a nucleic acid interface between proteins and the cell surface


(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).

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.

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..

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.

Initial experiment

We'll attempt to build an adaptamer that can bind thrombin and streptavidin simultaneously. More details to come. Oligos to be used here

Major Questions/concerns

  1. 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.)
  2. 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.
  3. 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?
  4. 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.
  5. 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.

Other projects

A couple of related projects that we haven't really researched..

  1. Having E. coli export an aptamer outside the cell
  2. Using aptamers to actually stimulate a response on the cell surface


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.

General Fusion

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All Medline abstracts: PubMed HubMed

E. coli cell surface display

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All Medline abstracts: PubMed HubMed

Notes on Autodisplay review (display1) 16369779

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:

  • Utilizes E.coli-native AIDA-I as scaffold
  • Detection: monoclonal antibodies and protease cleavage sites created
  • Protein unfolded during transport to cell surface.
  • Variety of proteins have been displayed (p.610)
  • Possibly to display catalytically active enzymes.
  • Dimerization of proteins has been observed! (unique to this system); work on tetramers in progress. Protein anchor floats around membrane.
  • Many proteins displayed: ~10^5 without loss of cell viability

The writer claims that the system is superior compared to all other display systems, but he might be biased since he created it.

Notes on expressing genes in different compartments review (display2) 11024362

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.

  • 5 options; a good summary is found on page 3.
    • Porins- Insert protein <= 60 residues
    • Fimbriae- Insert protein <= 15 residues
    • Lipoproteins
    • GPI anchor
    • Beta-autotransporter - same system as that used in display1
  • 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.

GPI anchor(display3) 9624691

Functional levansucrase bound to a GPI anchor, Inp, was expressed on the E. coli cell surface. Using these cells, they successfully converted sucrose to levan. Levansucrase, is about 400 a.a. residues long.

More bacterial display (display4) 12039531

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.

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.

Aptamers and Adaptamers

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All Medline abstracts: PubMed HubMed

Quantum dots conjugated to aptamers (apt1) 15541352 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...

Adaptamers (apt3) 12000850 This paper is also listed in the streptavidin section. See "Notes" under "Demonstrate Control.." project for a description.

CopA/CopT (apt4) 16199086 This recent review paper has a description of the CopA/CopT interaction which is utilized in apt3.

CopA/CopT (apt5) 10744017 This paper reveals the extremely intricate "deep-kissing" structure formed between CopA and CopT.

Thrombin (apt6) 1741036 Paper talking about selection for a DNA aptamer that binds thrombin. Seems like no one's evolved a better version yet: even recent papers talk about the same motif (16616893).

DNA aptamer to streptavidin (apt7) David Liu's paper about selecting for a DNA aptamer that binds streptavidin. The point here was using the nonhomolous random recombination instead of error-prone PCR to select for aptamers. The point for us is that it gives a DNA aptamer that binds streptavidin pretty well (40 bp motif kD ~100 nM).


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All Medline abstracts: PubMed HubMed

Display of Tetrameric streptavidin on B. subtilis spore surfaces (sa23) 16289701 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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All Medline abstracts: PubMed HubMed

Related articles

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Cd2+ binding (other1) 9893944 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.


Protein domain BioBricks presentation

Cell surface targeting

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