Team:Potsdam Bioware/Biobricks/All Biobricks

All Biobricks

As the project is divided into three subproject, the resulting biobricks are listet & characterized in the three categories. Furthermore the Biobricks for the new RFC "Potsdam Standard" are listed.
For detailed information on parts design, sequence, composites click the BioBrick numbers

Antibody Biobricks

All antibody module BioBricks for the smaller construct were designed out of already existing parts and BioBricks and were expanded with further functional units by assembly PCR. The advanced antibody construct was derived by de novo synthesis based on sequence information provided by GenBank and UniProt and by one already existing Biobrick part. The gene synthesis construct was optimized for expression in CHO cell and used as template for all subsequently derived BioBricks by PCR.

Mutation Biobricks

All the Mutation Biobricks are improved/modified versions of the wildtype AID that was available as a Biobrick ([http://partsregistry.org/Part:BBa_K103001 BBa_K103001]). AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.
Therefore this enzymes was used in our project for diversification of antibodies.With PCR and cloning we developed AID`s for the differnt requirements.

Characterization:

Mutation rate in CHO-cells:

Fig. 2: Comparison of the mutation rates between the wildtype AID, modified AID and modified AID-eGFP

We checked the mutation rate of wildtype AID([http://partsregistry.org/part:BBa_K929000 BBa_K929000]), modified AID([http://partsregistry.org/part:BBa_K929002 BBa_K929002]) and modified AID+eGFP([http://partsregistry.org/part:BBa_K929003 BBa_K929003]) (all expressed with CMV-promoter and hGHpolyA). Therefor we cotransfected CHO cells with a single chain construct and one of the AID versions. After certain expression time we purified the the single chain plasmids and transformed them into E.coli to enrich the mutated plasmids. After overnight culture and purification of the transformed plasmids, samples where sequenced.
There is a significant higher mutation rate when wildtype AID, modified AID or modified AID-eGFP is added, compared to the same experiment without AID. Surprisingly, the mutation rate of wt AID is two times higher than the mutation rate of the modified AID or modified AID-eGFP (Fig. 2). This observation is contrary to our expectation. The modified AID+eGFP is located in the nucleus (Fig. 3), that means our construct works and should have a higher mutation rate. One possible explanation is that the modified AID has a very high mutation rate and therefore the transfected cells die or inactivate the plasmid like it was observed for the wildtype AID (Martin and Scharff (2002)).

Green fluorescence reporter:
The modified AID was fused with eGFP to check 1.)the tranfection success 2.)the cellular lokalization of the improved AID and 3.) to select transfected cells via FACS.

• Transfection success and cellular localization:

Fig. 3: CHO cells were transfected A)with "modified AID+eGFP" (BBa_K929003)and B)with "modified AID+eGFP" (BBa_K929003) and a mCherry labeled nanobody construct (BBa_K929107)

The fluorescence microscope images(Fig. 3) show transfected cells A)with "modified AID+eGFP" (BBa_K929003)and B)with "modified AID+eGFP" (BBa_K929003) and a mCherry labeled nanobody construct (BBa_K929107)]]. "Modified AID+eGFP" (BBa_K929003)proved to be located in the nucleus. This shows that the deletion of the NES(Nuclear Export Sequence) and the addition of an NLS(Nuclear Localization Sequence) leads to an accumulation in the nucleus as it was planed. Therefore we would expect an increased mutation rate with "modified AID+eGFP" but as described above the experimental setup possible can not reflect this. It is also shown that transfected cells(green nucleus, Fig. 3 A&B) can be discriminated from not transfected (grey, Fig. 3). This can be used for a fast selection of transfected cells via FACS. Fig. 3 B shows that "modified AID+eGFP" can be used in combination with an other fluorescence labeld construct (mCherry labeled nanobody construct ,BBa_K929107) and co transfected cells can be easily found or selected via FACS.

Virus Biobricks

Potsdam Standard Biobricks