Team:Potsdam Bioware/Biobricks/All Biobricks

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Revision as of 20:00, 26 September 2012

All Biobricks

As the project is divided into three subproject, the resulting biobricks are listet in the three categories. Furthermore the Biobricks for the new RFC "Potsdam Standard" are listed.

Antibody Biobricks

Mutation Biobricks

All the Mutation Biobricks are improved/modified versions of the wildtype AID that was available as a Biobrick (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.

For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added. 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. For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added.

Characterization:

Virus Biobricks

Potsdam Standard Biobricks

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BBa_K929000 AID with CMV promoter and hGH-polyadenylation signal sequence	CMV-promoter & hGH polyadenylationsequence were added to the wildtype AID for strong expression.
BBa_K929001 modified_AID without NES, with NLS and Kozak sequence	To improve the mutation rate the AID was located into the nucleus, because there it mutates transcribed DNA. Therefor an aditional NLS(Nuclear Localization Sequence)was added and the naturally occurring NES(Nuclear Exprot Sequence) was removed.For stronger Expression an Kozak Sequence was added. To fuse modified AID((C-terminal of mod. AID) with RFC 25 parts, we added an AgeI restriction site.

@@ Line 9: / Line 9: @@
 ===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 genesWith PCR and cloning we developed AID`s for the differnt requirements.ID 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.
+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.<br>
+Therefore this enzymes was used in our project for diversification of antibodies.With PCR and cloning we developed AID`s for the differnt requirements.
-The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate.
+<table border=1>
+<tr><td>[http://partsregistry.org/Part:BBa_K929000 BBa_K929000] AID with CMV promoter and hGH-polyadenylation signal sequence</td>
+<td>
+CMV-promoter & hGH polyadenylationsequence were added to the wildtype AID for strong expression.
+</td>
+</tr>
+<tr><td>[http://partsregistry.org/Part:BBa_K929001 BBa_K929001] modified_AID without NES, with NLS and Kozak sequence</td>
+<td>
+To improve the mutation rate the AID was located into the nucleus, because there it mutates transcribed DNA. Therefor an aditional NLS(Nuclear Localization Sequence)was added and the naturally occurring NES(Nuclear Exprot Sequence) was removed.For stronger Expression an Kozak Sequence was added. To fuse modified AID((C-terminal of mod. AID) with RFC 25 parts, we added an AgeI restriction site.
+</td>
+</tr>
+For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added.
+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.
+For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added.
+====Characterization:====
 ===Virus Biobricks===