Overview

Our product is an anti-paclitaxel (ptx) antibody (33D9) that binds Antibody-drug conjugate coupled to ptx with high specificity and affinity (52B8hG1ptx). The most classical antibody production method is the hybridoma technique. Through preliminary laboratory research, we obtained the heavy chain variable region and light chain variable region sequences of 33D9, respectively. The primary objective of this experiment is to utilize molecular cloning technology to merge the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) found on pEE12.4 and pEE6.4, respectively. This combination aims to generate an expression vector capable of producing the complete antibody.

Table 1. Parts Collection for Contribution

Parts No.	Parts Name	Parts assembled	Part type	Contribution Type
BBa_K4961000	PEE12.4	basic part	Plasmid_Backbone	New part
BBa_K4961001	PEE6.4	basic part	Plasmid_Backbone	New part
BBa_K4961002	33D9-Light chain	basic part	Coding	New part
BBa_K4961003	33D9-Heavy chain	basic part	Coding	New part
BBa_K4961004	PEE12.4-33D9-H	composite part	Plasmid	New part
BBa_K4961005	PEE6.4-33D9-L	composite part	Plasmid	New part

Create a New Basic Part： BBa_K4961000, PEE12.4

BBa_K4961000 is a plasmid backbone called PEE12.4. The PEE12.4 vector is a Glutamine Synthetase (GS) gene expression system vector from PLonza Biotech. The target gene can be expressed at a high level in mammalian cells by using glutamine synthase to screen markers.

Create a New Basic Part： BBa_K4961001, PEE6.4

BBa_K4961001 is a plasmid backbone called PEE6.4. The PEE6.4 vector is a Glutamine Synthetase (GS) gene expression system vector from PLonza Biotech. The target gene can be expressed at a high level in mammalian cells by using glutamine synthase to screen markers. This is a commercial carrier, and the project designer's laboratory has made some improvements for subsequent experiments.

Create a New Basic Part： BBa_K4961002, 33D9-Light chain.

BBa_K4861002 is a coding sequence of the 33D9-Light chain. Through preliminary laboratory research, we obtained the heavy chain variable region and light chain variable region sequences of 33D9, respectively. This part of the sequence encodes the light chain of the antibody and can bind to the heavy chain to form a complete antibody.

Create a New Basic Part： BBa_K4961003, 33D9-Heavy chain.

BBa_K4861003 is a coding sequence of 33D9- Heavy chain. Through preliminary laboratory research, we obtained the heavy chain variable region and light chain variable region sequences of 33D9, respectively. This sequence encodes the heavy chain of the antibody and can bind to the light chain to form a complete antibody.

Create a New Composite Part: BBa_K4961004, PEE12.4-33D9-H.

The main task of this experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning technology to form an expression vector that can fully express the antibody. To construct our complete antibody expression plasmid, we asked the Gene Synthesis company to synthesize the DNA fragments, then, using the technique of molecular cloning(homologous recombination), we inserted the heavy chain DNA fragment of the antibody into the pEE12.4 vector. After obtaining the recombinant plasmid, we selected the PCR-correct monoclonal colonies and sent them to the company for sequencing. The validation results showed that we obtained the correct recombinant plasmid.

Create a New Composite Part: BBa_K4961005, PEE12.4-33D9-L.

The main task of this experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning technology to form an expression vector that can fully express the antibody. To construct our complete antibody expression plasmid, we asked the Gene Synthesis company to synthesise the DNA fragments, then, using the technique of molecular cloning(homologous recombination), we inserted the light chain DNA fragment of the antibody into the pEE6.4 vector. After obtaining the recombinant plasmid, we selected the PCR-correct monoclonal colonies and sent them to the company for sequencing. The validation results showed that we obtained the correct recombinant plasmid.