Contribution

Overview

This year, GreatBay_SCIE sets out to construct a cellulosome complex in order to degrade cellulose and PET present in fabrics at a faster rate. Cellulosomes are multi-enzyme complexes consisting of a scaffolding protein and two types of complementary recognition modules known as “dockerin” and “cohesin”. The cellulosome allows enzymes to work synergistically, which makes cellulosomes more efficient in degrading cellulose than any other free enzyme system. We aim to express cellulases, namely endoglucanase (TrEGIII), exoglucanase (CBHII), beta-glucosidase (NpaBGS), cellulase boosters (TaLPMO and MtCDH), and scaffold proteins (CipA and OlpB) with E.coli and Kluyveromyces marxianus.

To achieve the goal of degrading a more comprehensive range of materials in clothes, we successfully expressed PETase. For the future, we planned to fuse PETase with a dockerin protein so that PETase could bind to our scaffold, which would improve the efficiency of PETase.

We also express ferritin in our host E.coli so that we can recycle the cellulosome complex and thus be more sustainable and cost-effective. It is noticeable that we have come up with an original approach to verifying ferritin expression.

Here are the contributions we have achieved:

A New Approach for Verifying The Expression of Ferritin

We have invented a new approach for verifying the expression of ferritin by using RFP as a reporter molecule. This method is capable of assisting any future teams who intend to prove the production of ferritin.

The gene coding for RFP was fused with a plasmid that provided resistance to Chloramphenicol, while the gene coding for ferritin was connected to a plasmid that provided resistance to Kanamycin. We transformed the two plasmids coding for RFP and ferritin to DH5α.

A solid medium with both Chloramphenicol and Kanamycin was used to culture the DH5α so that we could select the DH5α containing both plasmids. The DH5α was transferred into conical flasks for enlarged cultivation. When the OD reached 0.1, we induced the expression of RFP with rhamnose. The culture was left to grow until the OD reached 0.6, at which we induced the expression of ferritin with IPTG and added Mohr salt as an iron source. Then, we resuspended the DH5α with tris HCL and placed the DH5α into Petri dishes with strong magnets.

After leaving DH5α with the magnets overnight, we placed the Petri dish under a blue light and saw through a filter that filtered out blue and purple light. We found that the DH5α, which emitted red light, was concentrated around the magnet, showing that ferritin was successfully expressed. We found that a high rhamnose concentration allowed better observation since it induced more RFP.

Figure 1. Schematic representation of the plasmid coding for RFP and the plasmid coding for ferritin in E.coli DH5α

Refinement of The Yeast Toolkit for Kluyveromyces marxianus

We refined the yeast toolkit by making the commonly used yeast toolkit available for Kluyveromyces marxianus.

Kluyveromyces marxianus (K. marxianus) is a non-conventional yeast that is not widely studied. However, its physiological and metabolic characteristics show great potential in biotechnological applications. Therefore, a more comprehensive yeast toolkit for K. marxianus is of significant importance.

We expanded the yeast toolkit proposed by Michael E. Lee. The toolkit was designed for Saccharomyces cerevisiae. To make the parts compatible with K. marxianus, we designed the Lac4 vector, which contained a pKmK.C1 origin, KanR selection marker, and Lac4 promoter, that could be successfully expressed in K. marxianus. We changed the origin into the pKmK.C1 origin for K. marxianus. This allowed the vector plasmid to be replicated within K. marxianus. We used KanR as the selection marker for K. marxianus and proved that it was an effective selection marker for K. marxianus.

In addition, we changed the alpha factor pre-pro secretion leader (Mα) for S. cerevisiae to the Mα for K. marxianus. With the help of the Mα for K. marxianus, we were able to directly extract the desired proteins from the supernatant. This eliminated the need for performing cell lysis, thus making the production of cellulases more cost-effective and convenient.

Figure 2. The Yeast Toolkit [1]

Using eforRed as A Reporter Molecule

We have developed a novel detection experiment for the interaction between cohesin I and dockerin I, and cohesin II and dockerin II, using eforRED as a reporter molecule.

We constructed several E.coli expression vectors for type I dockerin domain ligated with an eforRed domain, type II dockerin domain ligated with an eforRed domain, antigen domain ligated with an eforRed domain, respectively, and E.coli expression vector ligated with an eforRed protein. The successful binding of cohesins to dockerins was indicated by the red light emitted by eforRed. When type I dockerin with eforRed binds to the type I cohesins without eforRed, the residue displayed a red light after centrifugation. When the binding of eforRed-containing type I dockerin and the non-eforRed-containing cohesin was unsuccessful, the residue did not demonstrate the red light as the eforRed-ligated type I cohesins were left in the supernatant. The same happened to the type II dockerins ligated with eforRed and the type II cohesins without eforRed. This allowed us to visualize and verify the binding of cohesins to dockerins. This approach is helpful to any future teams who intend to test for the interaction between cohesins and dockerins (view the engineering success page for more details).

Reference

[1] Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.” ACS Synthetic Biology, vol. 4, no. 9, May 2015, pp. 975–986, 10.1021/sb500366v.