The dhdR gene was successfully cloned into an E. coli expression vector for subsequent production of purified DhdR protein. See the protein production and purification here and the corresponding part BBa_K4768000.

Expected sizes of the amplicons:

1. • The Histag-dhdR fragment was PCR amplified from the corresponding IDT gBlock with primers dhdR-pET21-F and dhdR-pET21-R. pET21 was linearized by PCR with primers pET19T7-F and pET19T7-R from a template miniprep. After PCR, the plasmid DNA PCR product was further digested by DpnI treatment.

2. • The amplification products sizes were checked by electrophoresis in an EtBr stained agarose gel. Figure 1A shows amplification products matching with expected sizes.

3. • PCR clean-up was performed on the amplification products.

4. • The Histag-dhdR fragment was inserted into pET21 by In-Fusion. Transformants were selected on ampicillin plates.

5. • 24 transformants were screened by colony PCR with primers flanking the insertion site within pET21 (dhdR-pET21-F and dhdR-pET21-R). 3 positive transformants (clones 4, 6 and 7) were detected (figure 1B).

6. • Plasmids were extracted from clones 4, 6 and 7 and digested by restriction enzymes (EcoRV and NdeI) to check the correctness of the assembly. Figure 1C shows correct restriction maps for clones 4, 6 and 7.

7. • Plasmids from clones 4, 6 and 7 were sent to Eurofins Genomics to check the insert sequence and flanking regions by Sanger sequencing. No mutations were detected in the insert sequence.

Figure 1: construction of pET21_dhdR. (A) Migration of the PCR products generated from the gBlock and pET21 plasmid onto a 0.8% agarose gel and EtBr staining. T- refers to the negative control (without DNA matrix). (B) Shows positive clones from the colony PCR screening. (C) Double and single digestion of the pET21_dhdR extracted from clone 4 by EcoRV and Nde I. The expected sizes are indicated.

The following construct was successfully cloned. It was used as a DNA template in PURE system for the expression of the thymidine phosphorylase enzyme in the presence of 2-HG. See the corresponding part BBa_K4768003.

Expected sizes of the amplicons:

1. • The dhdO-tymp fragment was PCR amplified from the corresponding IDT gBlock with primers T7-term-F and T7-prom-R. The pET21 plasmid was linearized by PCR from a template miniprep using the same primers as those for the pET21_dhdR construct. After PCR, the plasmid DNA PCR product was further digested by DpnI treatment.

2. • The amplification products sizes were checked by electrophoresis in an EtBr stained agarose gel. Figure 3A shows amplification products matching with expected sizes.

3. • PCR clean-up was performed on the amplification products.

4. • The dhdO-tymp fragment was inserted into pET21 by In-Fusion. Transformants were selected on ampicillin plates.

5. • 24 transformants were screened by colony PCR with primers flanking the insertion site within pET21 (T7-term-F and T7-prom-R). 11 positive transformants (clones 1, 2, 4, 10, 12, 15, 18, 19, 21, 22 and 24) were detected (figure 3B).

6. • Plasmids were extracted from clones 10, 19 and 21 and digested by restriction enzymes (EcoRV and BamHI) to check the correctness of the assembly. Figure 3C shows correct restriction maps for clones 10, 19 and 21.

7. • Plasmids from clones 10 and 21 were sent to Eurofins Genomics to check the insert sequence and flanking regions by Sanger sequencing. No mutations were detected in the insert sequence.

Figure 3: construction of pET21_tymp. (A) Migration of the PCR products generated from the gBlock and pET21 plasmid onto a 0.8% agarose gel and EtBr revelation. (B) Shows positive clones from the colony PCR screening. T+ and T- refer to positive control (gBlock amplification) and negative control (without DNA matrix) respectively. (C) Double and single digestion of the pET21_tymp extract from clone 4 by EcoRV and BamHI.

We attempted to prepare the following construct for converting the detection of HER2 into transcriptional activation of the sfgfp or tymp genes in PURE system. In parallel, the recombinant protein was produced in PURE system with PCR products from the gBlock as it already contains the SP6 promoter, an RBS and a T7 Terminator. See the cell-free experimentation results.

Expected sizes of the amplicons:

1. • The SP6Promoter-T7Nterm-SolubleLinker-Pertuzumab-TerminatorT7 fragment was PCR amplified from the corresponding IDT gBlock (with primers T7-F and T7-R). The pET21 plasmid was linearized by PCR from a template miniprep. After PCR, the plasmid DNA PR product was further digested by DpnI treatment.

2. • The amplification products sizes were checked by electrophoresis on an EtBr stained agarose gel. Figure 5A shows amplification products matching with expected sizes.

3. • PCR clean-up was performed on the amplification products.

4. • The SP6Promoter-T7Nterm-SolubleLinker-Pertuzumab-TerminatorT7 fragment was inserted into pET21 by In-Fusion. Transformants were selected on ampicillin plates.

5. • 16 transformants were screened by colony PCR with primers flanking the insertion site within pET21 (T7-F and T7-R). 1 positive transformant was detected. (figure 5B).

6. • Plasmid was extracted from clones 8, 6 and 15 and digested by the restriction enzyme BsaI (clone 8) and EcoRV and XhoI (clones 6 and 15) to check the correctness of the assembly. Figure 5C shows correct restriction maps for clone 8.

7. • Plasmid from the clone 8 were sent to Eurofins Genomics to check the inserted sequences.

8. • The sequencing results obtained after we performed these experiments showed a 675-nucleotide deletion within the region of the heavy chain of Pertuzumab. However, the production of Pertuzumab-SL-T7Nterm we performed in parallel indicated the production of a protein with the expected size of 69 KDa. It remains to be investigated whether this deletion originated during the PCR that preceded the cloning or at the sequencing stage.

Figure 5: construction of pET21_Pertuzumab-SL-T7Nterm. (A) Migration of the PCR product generated from the gBlock and (B) pET21 onto a 0.8% agarose gel and EtBR revelation. (B) shows the PCR screening. T+ and T- refer to positive control (gBlock amplification) and negative control (without DNA matrix) respectively. (C) Represents the double digestion of plasmids extracted from clone 8, 6 and 15 and digested by Bsa (clone 8) and EcoRV and XhoI (clones 6 and 15).

We attempted to prepare the following construct for converting the detection of HER2 into transcriptional activation of the sfgfp or tymp genes in PURE system. Switching the vector didn’t allow to succeed with the In-Fusion step. Overlap PCR was attempted once again.

Expected sizes of the amplicons:

1. • The P6Promoter-Trastuzumab-SL and the T7Cterm-TerminatorT7 fragments were PCR amplified from the corresponding IDT gBlocks (with primers T7-F and Trastuzumab-R1’ for SP6Promoter-Trastuzumab-SL, and Trastuzumab-F1’ and T7-R for T7Cterm-Terminator T7). The pET21 plasmid was linearized by PCR from a template miniprep. After PCR, the plasmid DNA amplification product was further digested by DpnI treatment.

2. • The amplification products sizes were checked by electrophoresis on an EtBr stained agarose gel. Figure 9A and 9B shows amplification products matching with expected sizes.

3. • After gel extraction of the insert and PCR clean-up of the linearized plasmid, the SP6Promoter-Trastuzumab-SL and T7Cterm-TerminatorT7 fragments were inserted into pET21 by In-Fusion. Transformants were selected on ampicillin plates.

4. • 24 transformants were screened by colony PCR with primers flanking the insertion site within pET21 (T7-F and T7-R). No positive transformant was detected (figure 9C).

5. • The lack of positive strain very probably results from a failed In-Fusion of the 2 inserts containing Trastuzumab and splitT7 and the linearized pET21.

6. • After the failure of the In-Fusion we tried to merge the 2 fragments together by PCR using the primer T7-F and T7-R. We used our first PCR products of the 2 fragments as a matrix.
   We obtained 4 amplification products and none corresponds to the weight expected (4031 bp) (figure 10).

7. • With these results we supposed that there is a problem of hybridization with the primers T7-F and T7-R. We decided to try another In-Fusion and to use the primers pET21seq-F and pET21seq-R which are on the plasmid for the PCR screening.
   We obtained an amplification product of approximately 600 bp corresponding to pET21 (figure 11).

8. • As done with SP6Promoter-T7Nterm-SolubleLinker-Pertuzumab-TerminatorT7, we decided to switch to pET21 as a cloning vector in order to obtain a better assembly.

9. • All the clones screened with these primers were transformed with the native plasmid.

Figure 9: construction of pET21_Trastuzumab-SL-T7Cterm. (A) & (B) Migration of the PCR product generated from the gBlock and pET21 onto a 0.8% agarose gel and EtBR revelation. (C) colony PCR screening.

Figure 10: Migration of overlap PCR product of gBlocks and pET21 onto a 0.8% agarose gel and EtBr staining.

Figure11: colony PCR screening using the primers pET21seq-F and pET21seq-R.