1. Constructing our expression vector with Dockerin
This year our team aims to build a new pathway with two enzymes in E.coli. For improving the reaction efficiency, we use a powerful tool—Synthetic Protein Scaffolds. The protein scaffold is an assembly platform and it could bring enzymes with matching dockerin together so that reaction efficiencies can be optimized through the passing on of intermediates between co-located enzymes.
So the first step in our project is to insert the sequence of dockerin behind gene which encodes target enzyme (Figure1). We used a high-efficiency method—one step cloning to do this work. In addition to dockerin, we also add a His tag at the 5’ end of dockerin, so that we could easily purify our fused protein to do in vitro experiments.
Figure1. Dockerin is inserted behind our gene of interest and His tag is added at the 3’ end for purification.
2. Testing Scaffold Protein System
Before doing our experiment in vitro, we tested the scaffold to ensure that it is functional. We used a Synthetic Protein Scaffold with a cellulose-binding domain. This is to say, when we mix enzyme with dockerin and scaffold protein, we can use cellulose to purify this enzyme-scaffold complex. Then through SDS-PAGE, three kinds of protein (scaffold, BFD and TalB) can be detected. If enzyme with dockerin couldn’t bind with scaffold, through this purification way, we can only detect scaffold protein.
3. Testing pathway in Vitro
To verify that our designed pathway is feasible, we first do the experiment in tube. Firstly, we use His tag to purify BFD-CtDock and TalB-CcsDock. Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli . Secondly, we add BFD-CtDock into reaction tube with its substrate—Formaldehyde. After BFD catalyzes formaldehyde to intermediate— Glycolaldehyde and DHA, we add TalB-CcsDock into the reaction tube to synthesize xylulose. Finally, we detect xylulose through High Performance Liquid Chromatography (HPLC). HPLC is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). The components of the sample move through the column at different velocities, which is a function of specific physical interactions with the adsorbent. The time at which a specific analyte elutes (emerges from the column) is called the retention time. The retention time measured under particular conditions is an identifying characteristic of a given analyte. The data obtained from HPLC will be compared to standard product’s data to determine the presence or absence of the target product.
Figure2. Flowchart of HPLC in analyzing a sample.(http://laboratoryinfo.com/hplc/)
We also use Synthetic Protein Scaffold to combine BFD and TalB. The HPLC results showed that reaction efficiency is greatly improved.
4. Testing pathway in Vivo
After in vitro experiment, we try to verify that our designed pathway is feasible in vivo. We transformed pET28a-BFD and pET22b-TalB into E.coli. We first add E.coli expressing BFD-CtDock into reaction tube with formaldehyde. When the reaction is over, we add E.coli expressing TalB-CcsDock into the tube to synthesize D-xylulose. The target product is detected by HPLC.
In order to reduce the in and out of intermediates among different E.coli, we constructed a new plasmid pET28a-BTT(BFD-TalB-TalB) to make the whole pathway possible in one cell. And in consideration of TalB’s low catalytic efficiency compared with BFD, we inserted two copy of TalB’s sequence in this new plasmid(Figure3). We add the new type of E.coli into reaction tube with formaldehyde, and detect D-xylulose by HPLC.