*prof. Jacek Bielecki
*prof. Jacek Bielecki
*dr. Radoslaw Stachowiak
*dr. Radoslaw Stachowiak
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== Our Project ==
== Our Project ==
Monoclonal Antibodies === |+|
|−|Monoclonal antibodies are proteins produced by cells of the immune system for defense against pathogens. Because of high target recognition specificity antibodies are object of research in many laboratories and pharmaceutical companies around the world. Currently exist numerous therapies based on monoclonal antibodies for diseases such as cancer, allergy, viral infections and autoimmunological diseases. Antibodies are also important research tool used to identify various chemical and biological particles. |+|
of . of in the . to . > and of . in to . In this case used to . will . cell surface to survival of of . will be to and . will be culture medium. will allow to .. and .
|−|<div> Unfortunately traditional methods of making antibodies are complicated, expensive, time and work consuming because they are based on immunization of mammals. It is worth to mention that there is constant trend to reduce usage of laboratory animals in scientific experiments, mainly for ethical reasons. Because of that there is constant effort to develop various techniques allowing antibody production in yeast or bacterial cells. In this case only small fragments of antibody are used . Those fragments retain ability of binding to target antigen in the same manner as full antibody does. Currently existing methods are still very time consuming and require large amount of laboratory work to develop single antibody.</div><div>Technique proposed by our team will allow easy development and production of monoclonal antibodies in bacterial cells.</div> | |
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|−|=== Antibody producing GEM === | |
|−|Our research project is divided in three basic stages: | |
|−|*In stage 1 we will introduce mechanism of targeted increase of genetical variability in regions of bacterial genome encoding antibodies. In this situation every bacterial cell in population will express different antibody on its surface . | |
|−|*Goal of stage 2 is to provide selection mechanism, which will cause survival of the cells only when expressed antibody binds with target protein. Selection mechanism is based on antibiotic resistance induced by binding of target protein to antibody on cell surface. | |
|−|*In stage 3 cellular metabolism will be switched to efficient antibody production and export. <br>Antibody will be then purified from culture medium. At all stages we have planed various control experiments, which will allow us to determine our progress. | |
|−|<br>This is very simplified description of our ‘biological machine’. Success of our project would allow simple, fast and cheap production of antibodies with desired specificity. | |
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[[IGEM:UW/2008/Notebook/iGEM_UW_Team | Our Lab Calendar and Notebook]] == |+|
Revision as of 09:56, 30 June 2008
University of Warsaw IGEM 2008 page
Welcome to University of Warsaw IGEM group page for year 2008
DNA Sequencing and Synthyesis
Faculty of Biology, University of Warsaw
Foundation for Polish Science
Warsaw University Consultative Council for Students' Scientific Movement
- prof. Jacek Bielecki
- dr. Radoslaw Stachowiak
Our team is planning to develop two connected devices. The first device will introduce mutations to defined region of plasmid in E. coli cells (level of mutations in the rest of bacterial genome should be near to background). We’ll try to achieve this using human protein AID (Activation Induced Deamidase). It acts on single stranded DNA causing C --> U substitutions. This activity causes point mutations and activation of various DNA repair systems potentially including error-prone DNA repair systems. Site-specific in vivo DNA mutagenesis device can be used to produce proteins with new activities. In this case proper selection pressure must be applied. Our second device will be used to create proper selection conditions. It will use beta-lactamase split into two complementing domains anchored on E. coli cell surface and culture medium containing ampicillin to promote survival of clones expressing desired phenotype and ensure death of others. One beta-lactamase domain will be bound to cell membrane and protein which interactors we will be looking for. The other domain will be connected with ligand and added to culture medium. Interaction between protein and ligand will cause complementation of beta-lactamase domains and ampicillin-resistant phenotype. Connection of those two devices may allow to build machine screening for strongly interacting pairs of proteins i.e. antibody and antigen.
Full project description
Out first device will introduce mutations to the specific fragment of plasmid in E. coli cells. We want to limit mutagenesis to particular sequence and avoid mutations in bacterial chromosome. The basic part of this device is human protein AID, which activity is crucial for maturation of proteins in lymphocytes. AID acts on single-stranded DNA during transcription causing conversion of cytosine to uracil. This leads to point mutations. Aid gene has been already expressed in E. coli and caused increased mutation rate of some highly transcribed loci.
The first construct we want to prepare – the A plasmid will encode aid gene under arabinose-inducible promoter (PBAD). AID expressed from this plasmid should act on highly transcribed loci. Sequences expressed from T7 promoter have one of the highest transcription levels. That’s why our next construct will carry transcription fusion between AID and T7 polymerase (plasmid A=T7). Some authors suggest that AID may act as oligomer, so we will create construct encoding transcription fusion between AID and AID-T7 translation fusion (plasmid A+A=T7). In that case fusion protein AID-polT7 may recruit free AID. Control plasmid T7, carrying only T7 polymerase gene, will be also created.
To test usefulness of constructs containing various variants of AID protein some reporter system must be created. In our case it will be based on pZC plasmid - the one-copy minireplicon of F plasmid. pZC encodes alpha fragment of beta-galactosidase under two promoters (T7 and lac). using pZC we will be able to assess mutation level of sequence encoding reporter gene (mutations will result in white colonies). We expect mostly C T substitutions but we hope that error-prone DNA repair systems will be activated causing insertion of random nucleotide into mutation site. Results of blue-white mutation screening will be confirmed by sequencing of reporter gene. We also have to determine level of mutations occurring on chromosome. One of the simplest tests is to determine the frequency of mutations resulting in rifampicin-resistant phenotype. Optimal culture and induction of mutator system genes expression conditions need to be found. If level of mutations will be to low we’ll use bacterial strains with damaged DNA repair systems.
The task of our second device is to select proteins interacting on the surface of E. coli cells. Selection will be based on complementation of beta-lactamase fragments. Complementation occurs only when the fragments are in close proximity and it is not spontaneous. Connecting beta-lactamase fragments to two interacting proteins will allow complementation and will make cells ampicillin resistant. We want to connect one beta-lactamase fragment to outer membrane anchor protein and add the other fragment connected with ligand protein to culture medium. In ampicillin containing medium cells having protein that stronger binds to ligand will have selection advantage over others. It will allow screening of libraries encoding antibody fragments or receptor proteins to select best binding clones for specific ligand. But our main aim is connecting this device with previously described one to create variance and simultaneously select cells expressing desired phenotype i.e. cells expressing antibody fragments with high affinity to given antigen.
On this stage numerous control and test constructs will be created and optimal screening device will be selected. The basic part of most constructs will be outer membrane anchor from OmpA protein. Other parts include alpha and omega beta-lactamase fragments and A and Z proteins. A and Z are small strongly interacting proteins. Usage of A and Z protein fused with receptor and ligand protein will ensure their close proximity and allow close contact of beta-lactamase fragments. Interaction of A and Z proteins will also help us in assessing ampicillin resistance level given by two complementing beta-lactamase fragments. In final stage of our project constructs for both receptor and ligand protein overexpression and purification will be created.