Team:Potsdam Bioware/Project/At a Glance

At a Glance

Antibody Generation System

The main goal of our antibody generation system is to produce high affine antibody using an antibody module, a mutation module and a selection module to ensure that the cells which express a high affine antibody survive.
For the antibody module, we transiently and stable transfected CHO cells with two antibody constructs. The first one consists of a single chain against the epidermale growth factor receptor domain three, a transmembrane region and a signal peptide. The latter ones 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 with the same function like above and a switchable region to ensure the shifting from membrane standing to soluble state of the antibodies.
The mutation module consists of one key enzyme, the activation induced cytidine deaminase (AID). This enzyme is commonly used in mammalian immune systems to induced the hypermuation and thus antibody maturation in activated B-lymphocytes. We used the wildtype form and a modified variant of this enzyme with a nuclear localization sequence and without a nuclear export sequence for transfection in CHO cells to induce hypermutation. The transfected AID induce hypermutation in antibody transfected CHO cells and thus change the antibody binding regions stochastically.
To select CHO cells which produce a high affine antibody, we constructed a 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 effectively. Consequently, only the CHO cells survive which produce a high affine antibody mutated by the AID.

Collaboration

We are working together with the University Freiburg team to test their Transactivator-like (TAL) proteins by sending one of our modified AID enzymes for ligation to the TAL domain. Team Freiburg, on the other hand, plans to send back the TAL domain linked to the AID via glycine-serine linker. Afterwards, we aim to check the mutation rate of the specifically directed AID to our antibody sequence.

SocialBrick

The idea of SocialBrick is to divide all human practice acts into different parts: the SocialBricks. Here, the SocialBricks stands for every activity which aims to inform the people about the Synthetic Biology. We hope that the term SocialBrick 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.
The main results of the human practice this year are 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 by using the classical way 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 4’ 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.