The whole project in general included 3 parts, the literature review, cloning and assays. The assays were further divided into 5 specific goal, which were the confirmation of protein expression in the produced clones, the measurement of codon optimization effectiveness, testify the generation of eforRed signal and the drug and DSF treatment assays.

Literature review could help us in the planning of the whole project. Details like the protein tag for purification, cell culturing, gene expression condition, gene sequence variation in different bacterial strains were the main objectives in this step. Besides, codon optimization was also included in this planning process. As we knew that different organism strains would have different preferences in codon translation, we did codon optimization in the online database system in order to design the gene sequences which were the most suitable to be expressed in E. Coli.

Figure 1: Cloning stages of the project. Stage 1 involves 6 separate inserts. Stage 2 and 3 involves pair of inserts in each plasmid, named as the construct A, B and C. Stage 4 and 5 involves the ligation of the 3 constructs to the same plasmid.

Figure 2: Stage 2 involves the irreversible ligation of insert pairs by the restriction site XbaI and SpeI. As XbaI and SpeI have the same sticky end, they can be ligated with each other. Once ligation is done, the site cannot be neither recognised by XbaI enzyme nor SpeI enzyme.

The next step in our project was the cloning (figure 1). The biobrick cloning involved 5 stages as shown in the figure. Overall, we had to insert 6 biobricks into a plasmid for transformation, which was a difficult task to attain. To made sure the cloning progress could be monitored, we divided the biobricks to be cloned in pairs, which were called the “constructs” in our project description. A biobrick “construct” contained two gene inserts that were ligated by the XbaI and SpeI restriction sites, forming a scar which could not be digested anymore (figure 2). The formation of scar ensured that unnecessary restriction digestion would not happen in the further cloning stages.

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Figure 3: (a-c) The figures showed assays in th project.

The last step would be the assay part (figure 3). We would like to first ensure proteins involving in the intracellular signal transduction can be expressed. Therefore we did the western blots of RpfC, RpfG and Clp proteins. Next, testing the expression efficiency of codon-optimized RpfG was important to prove the success in our work. In this assay, RpfG of old sequence and of the optimized sequence were expressed in E. Coli. After confirming the signal transduction part expression, the next assay target would be the signaling protein, eforRed. Drug assays were to prove that whether the molecular pathway of this system works. The drugs used were azathioprine and sulfathiazole. Both of them were the c-di-GMP suppressive drug. In our project idea, we expected that after suppressing the c-di-GMP level, Clp protein would be free to bind DNA and promote gene transcription. The assay helped us to test the relationship between the c-di-GMP level, Clp DNA-binding affinity, and the eforRed expression. Last but not least, DSF assay was the final goal of the molecular aspect of the system. We aimed to modify the E. Coli and make it be able to detect and respond to DSF molecules in the culture environment. In this DSF assay, cells were treated with DSF in different concentration and were tested for the eforRed expression.

Lab manual

Team:Hong Kong-CUHK/Experiments

Experiments

Lab manual