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Team:Potsdam Bioware/Project/At a Glance
From 2012.igem.org
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At A Glance
Antibody Generation System
The goal of our "Antibody Generation System" is streamlining the generation and production of antibodies by integrating all steps in one cell line. Antibodies are indispensable tools for research and diagnostics and they are used in diverse applications such as ELISA, western blot, affinity purification, and imuno-histology. Antibodies also represent the most important class of biopharmaceuticals with US sales of over 18 billion US$ in 2010 (Aggarwal, 2011) and varied therapeutic areas such as cancer, arthritis, and macular degeneration. Depending on the use of the antibodies specific demands must be met. In research, the murine IgG is very typical. For therapy, antibodies need to be 'humanized' to avoid immunological reactions. For some applications downsized or modified versions of antibodies can by used such as single chain Fv fragments (scFv), Fab fragments, or heavy chain only (cameloid) antibodies. However most of the time, the Fc part of the antibody provides crucial functionality like secondary antibody binding in research or cell dependent cytotoxicity in therapy. Most antibodies produced in larger scale a recombinantely expressed in CHO cells, as CHO cells mimick human gylcosylation and can produce large amounts of antibodies. In general, almost 70% of all biopharmaceuticals are produced in CHO cells (CHO consortium, 2012).
So far, antibodies are typically produced by immunizing mice, sacrificing mice, generating hybridoma by cell fusion and selecting the desired hybridoma clones. This is very time consuming and expression quality varies widely. In addition, only natural murine antibodies can be obtained. For modifications or large scale production, the antibodies genes are mostly identified by phage display and then recloned in a defined expression cell line such as CHO. As this is very time consuming, E. coli based antibody fragment libraries and phage display emerged as a second route. However in this case only scFv or Fab are obtained in the first place and most applications benefit from the divalent character of the full antibody and need the Fc part. The Fc part requires glycosylation for stability requiring the move from E. coli to eukaryotic cells, which again is a laborious step.
to produce high affine antibodies using an antibody module, a mutation module and a selection module to ensure that the cells that express a high affine antibody survive.
For the antibody module, we transiently and stably transfected CHO cells with two antibody constructs. The first one is a single chain antibody against the epidermal growth factor receptor domain three. Additional transmembrane region and a signal peptide ensure that the construct is presented on the cell surface. The second one contains a nanobody against GFP, a Fc domain, a transmembrane region and a signal peptide that also direct the construct to the surface. A switchable region ensures the possibility of shifting from membrane standing to soluble state of the antibodies with help of Cre recombinase.
The mutation module consists of one key enzyme, the activation induced cytidine deaminase (AID). This enzyme is commonly used in mammalian immune systems to induce the hypermuation and thus antibody maturation in activated B-lymphocytes. We created the wildtype form and a modified variant of this enzyme that has a nuclear localization sequence and no nuclear export sequence. Both of them were used for transfection into CHO cells to induce hypermutation. The transfected AID induces hypermutation in antibody transfected CHO cells and thus change the antibody binding regions stochastically.
To select CHO cells which produce high affine antibodies, we designed the selection module. This module consists of viruses which show the corresponding antigen for the nanobody GFP on the surface by using a fusion protein. The virus has an antibiotic resistance cassette. By binding the high affine antibody with the surface presenting antigen the virus is able to infect the CHO cells efficiently. Consequently, the CHO cells only survive if they produce high affine antibodies mutated by the AID.
Aggarwal, S. (2011). What’s fueling the biotech engine-2010 to 2011. Nature biotechnology, 29(12), 1083–9. doi:10.1038/nbt.2060 CHO consortium http://hugroup.cems.umn.edu/CHO
SocialBricks
The idea of SocialBricks is to divide all human practice activities into different parts: the SocialBricks. Here, the SocialBricks stand for every activity which aims to inform people about the Synthetic Biology. We hope that the term SocialBricks will be accepted like the term BioBrick and will be integrated in a registry to show the society what every team has done for more elucidation.
For this year we focused on two SocialBricks: “Science meets Politics” and “Science meets People”. For the first one we interviewed politicians from the German parliament: the Bundestag and discussed about Synthetic Biology. For the second part we organized a survey to ask about people's knowledge of Synthetic Biology and for their opinion on this scientific field. We also organized the day of synthetic biology in Potsdam on the main street.
The main result of the human practice this year is that the citizens and the politicians see the great potential of Synthetic Biology but also the challenges of this new scientific field.
Potsdam Standard - a hybrid approach to assemble your gene of interest
The main problems in using the classical approach to assemble different parts with restriction enzymes are on one hand ineffective enzymes with different optimum conditions and on the other hand illegal restriction site in the sequence which restricts the use of standard assemblies.
That is the reason why we tried to establish a new RFC with a reduced use of restriction enzymes. This cloning standard is based on the use of thiophosphate primer at the 5’ end for PCR to amplifying the insert. The insert is incubated in iodine/ethanol solution to knock out the 5’ thiophosphates. After that, the new standard cloning vector, developed by us, with a RFP expression cassette as a ligation control is digested with the enzymes Apa I and Sph I. These enzymes generate 3’ overhangs which correspond to the 3’overhang generated by knocking out the thiophosphates. The digested backbone and the pliced insert is mixed, ligated and transformed into E.coli.
To proof the new assembly standard, we insert the AID into the new standard cloning vector using the Potsdam Standard. After sequencing, we saw that the cloning was successful without any mutation in the AID sequence.
Main Results
Antibody Module: Antibody Expression
Mutation Module: Antibody Maturation by Mutation with the AID Enzyme
Selection Module: Selection or Screening for the Desired Clone
Modeling: Analyzing and Predicting the Viral Antibody Selection
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Modeling is a powerful tool to understand complex interactions or processes. We created a model to illustrate our selection system and to get a better understanding of its behavior under different conditions. Thereby, we wanted to answer two main questions:
To answer these questions we did both deterministic and stochastic modeling using MATLAB. Furthermore, we did a huge parameter analysis for each model to check what kind of influence each parameter has on our selection system. Stochastic results showed that the selection is finished on random time points after 100 h or later (see Fig. X). Furthermore the analysis of initial concentrations of WT cell and virus revealed that there is an optimum for initial concentrations of WT cell and virus. Both to less and to excessive concentrations prevent the success of the selection system |
