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.

Characterization:

Expression of Smaller Construct in CHO Cells

Fig. 1: eYFP fluorescence of the small construct in stably transfected CHO cells

The small antibody construct was designed with a eYFP reporter to check the a) expression level of the construct, its b) cellular localization and to enable the selection of the transfected cells via FACS.

The fluorescence microscopy image (Fig. 1) shows the transfected CHO cells with the smaller antibody construct containing the scFV425 (BBa_K929101). It can be proven that the construct is expressed in CHO cells and also shows a high fluorescent signal. Its accumulation in the vesicular compartment of the golgi apparatus implements a membranous localization. FACS selection was successfully realized.

Expression of Advanced Construct in CHO Cells

Fig. 2: mCherry fluorescence of advanced antibody construct showing membrane localisation on CHO cells

The fluorescence microscopy image (Fig. 2) shows the transfected CHO cells with the advanced antibody construct containing the anti-GFP nanobody (PDB: 3OGO). The images indicate that the construct is expressed in CHO cells and shows a high fluorescent signal sufficient for detection. With fluorescence microscopy, confocal microscopy, immunfluorescence and FACS we were able to show the expression of the transfected parts. Under certain conditions we have seen a membrane localization of the advanced construct (Fig. 2) yet we have not been able to generate cells that transport enough molecules to their surface for successful detection.

Functionality of Applied Cre/LoxP System

Fig. 3:Western Blot of purified Nanobody-Fc from supernatant of cells co-transfected with Cre recombinase and advanced construct

As an additional functional unit the advanced construct contains two LoxP sites which flank the membrane anchoring region, thus allowing the switch from surface presentation to secretion on genetically level.

By co-transfecting CHO cells with our advanced antibody construct and the cre recombinase (pBS185) we were able to proof the functionality of the applied cre/LoxP system. The immune blot image (Fig. 3) shows purified Nanobody-Fc from supernatant obtained from co-transfected CHO cells colored with anti-human Fc coupled to streptavidin. Purification was carried out with magnetic beads. A correct band can be seen at 41kDa.

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.

Characterization:

Mutation rate in CHO-cells:

Fig. 4: Comparison of the mutation rates between the wildtype AID, modified AID and modified AID-eGFP

We checked the mutation rate of wildtype AID(BBa_K929000), modified AID(BBa_K929002) and modified AID+eGFP(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. 4). This observation is contrary to our expectation. The modified AID+eGFP is located in the nucleus (Fig. 5), 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. 5: 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. 5) 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. 5 A&B) can be discriminated from not transfected (grey, Fig. 5). This can be used for a fast selection of transfected cells via FACS. Fig. 5 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.

Selection BioBricks

Characterization:

Figure 6: expression of the 3rd EGFR domain in E. Coli detection was performed using SDS-PAGE and Western-blot

Using the Biobricks with the Sortase motif fused to the VP2/3 region BBa_K929200; BBa_K929200 it is easily to ligate any proteins containing the C-terminal sortase motif with the enzyme sortase. We were interested to bring EGFR on the surface of the virus. Therefore we expressed the 3rd EGFR domain with the C-terminal sortase motif BBa_K929202 in E. coli. We detect successful expression of EGFR on western blot (Fig. 6)

Potsdam Standard BioBricks

The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa et al. (2010), Liu XP et al. (2010)). The original PLICing is based on the amplification of insert and backbone to ligate them together by knocking out thiophosphates at the 3' end. For the Potsdam Standard, we designed a new cloning vector with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3. In the prefix we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.
The insert was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the new assembly vector. After that the digested vector and the PLICed insert were mixed and transformed into E. coli. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.

Characterization:

We tested different combination to prove the most efficient experimental design. Therefore, we tried four different conditions: the ligation of the pliced GOI with the unpurified digested backbone with and without ligase and the ligation of the pliced GOI with the purified digested backbone with and without ligase. After transformation, we picked only clones which have lost their red fluorescence indicating that the ligation was successful (fig. 9).
To quantify the results, we counted the red colonies and the colonies without red fluorescence (fig. 10). It is obvious, that the ligation with the electrophoretic purified digested backbone is more efficient than without any purification. Nevertheless, the use of ligase is not necessary for the ligation process under these conditions.

Fig. 7: Overnight culture of transformed E.coli	Fig. 8: After centrifugation, the cell pellets are coloured like cherries
Fig. 9: Results of different ligation conditions	Fig. 10: Results of different ligation conditions