The experimental part of our project consisted on implementing, or more precisely trying to implement, the basic design of the PLA system. Thus, we aimed to express PhaC, Pct and LDH genes in our host organism Pseudomonas putida. Before starting, we designed our genes and command them using the sponsoring of IDT gBlocks. We also asked for pSEVA plasmids, which were provided by Víctor de Lorenzo Lab (CNB-CSIC), and we designed primers necessary for cloning and checking PCR protocols.
Figure 1 shows a general schema on what we planned to do for PLA production.
A part from these experiments, we had to work parallely on the following points:
- Growth experiments: We wanted to check different growth conditions depending on carbon source on the medium.
- BioBrick construction: We had to clone our genes, pSEVA promoters and other potential devices on pSB1C3 as BioBricks and characterize them.
- Gene deletion: We wanted to try the gene deletion of the WT PhaC on P. putida genome, as it could help improving the yield.
- Others: We had to help Paris Bettencourt by running HPLC and MS experiments, and report growth conditions to Imperial College.
Figure 1. Experimental plan for PLA production.
The steps illustrated on the Figure 1 schema correspond to the following:
- First, we clone separately Pct and PhaC gBlocks into pSB1C3 by Standard BioBrick Assembly; we transform E. coli with the ligated plasmid and we do mini-prep. Then, we do BioBrick Assembly with the two plasmids constructed in order to insert PhaC gene into Pct plasmid and construct the operon; we do transformation in E. coli and mini-prep. Finally, we clone the operon into pSEVA224 and repeat transformation and mini-prep steps.
- Parallely, we clone LDH gBlock into pSEVA2311 by BioBrick assembly, we transform E. coli and we do mini-prep.
- We transform P. putida with our pSEVA2311 and pSEVA224, containing LDH and the PhaC-Pct operon respectively. We grow the transformed colonies and we induce gene expression (by IPTG and Cyclohexanone). We expect in this step to have PLA.
- We finally extract the PLA vesicles from inside bacteria cells and we do PLA characterization experiments.
Tasks and associated protocols
Once in the bench, we had to face the complexity and stochasticity of biology and we could not follow straight the steps presented. Thus, we splitted the potential tasks and tried to work on them parallely. Figure 2 shows the schema of the general tasks.
Figure 2. Schema for experimental tasks.