Engineering Success

Overview

To realize the goal of advancing the project smoothly, we follow the rule of “Design-Build-Test-Learn Cycle”. We have conducted two-cycles processes to move the project forward. In the first cycle, we overexpressed three genes related to manganese oxidation into plasmid, and finished building and testing the porduction of enzymes (Multicopper oxidase (MCO), Manganese catalase (MnC) and Manganese peroxidase (MnP)) in P. putida chassis. In the second cycle, we engineered the Cytochrome c maturation (Ccm) operon with a stronger promoter, achieved to express multiple enzymes plus corresponding manganese oxidation reaction in P. putida.

Figure 1: Engineering cycle diagram of our project

Cycle 1-Design

First, we decided to construct and validate three genes related to Mn oxidation in P. putida. Through gene synthesis, we obtained the target gene, cloned the genes of three related enzymes into pBBR1-MCS5 plasmid. Then transformed and tried to express the related enzymes in P. putida.

Cycle 1-Build

Figure 2: Colony PCR results for all our samples

Cycle 1-Test

Figure 3: PCR and SDS-PAGE protein check gel electrophoresis result of MCO, MnC and MnP.

                      Control                  MCO                 MnC                  MnP

Figure 4. The Mn oxidation induced by P. putida, which overexpresses MCO, MnC and MnP.

As can be seen in Figure 4, the overexpression of each of three enzymes did not achieve the desired manganese oxidation results.

Cycle 2-Learn

Because the achieved results is far from satisfatory, we need to find a more efficient way to upregulate the Mn oxidation, thus improving the degradation of lignin. Through learning from literatures and discussions with team advisorss, we learned that the production of enzymes can be achieved by genetic engineering of the promoter of Ccm operon.The promoter can simultaneously regulate the expression of different Ccm enzymes (ccmA, ccmB, ccmC, ccmD, ccmE, ccmF, ccmG, ccmH and ccmI). Therefore, we decided to replaced the promoter of Ccm operon with a stronger one, as shown in our design disgram.

Cycle 2-Design

We use a stronger promoter (part code: BBa_5204002), which is reported to be a stronger promotor of P. putida, to replace the original promoter of Ccm operon. Then we constructed the vectorin P. putida In addition,gentamicin was added to all media to ensure that the surviving cells contained our construction plasmid.

Figure 5. Schematic representation of the ccm operon of Pseudomonas putida.

Cycle 2-Build

Figure 6. Promoter modification engineering to improve the transcript level of the ccm operon.

Cycle 2-Test

Figure 7: QPCR results of ccmE and ccmL, and the Mn oxidation results induced by P. putida.