Introduction

The aim of our project was to obtain a transformed bacterial population containing both overexpressed glycogenosynthetic and cellulolytic genes for the final starch-like production.

Results

For this, in the first instance, gene constructs based on the Part Registry were developed computationally, with subsequent modification and optimization to avoid restriction targets to be used within the insert.

The first step of the experimental design is the digestion of the inserts and their ligation into individual plasmids for characterization. As can be seen in the agarose electrophoresis gels, the digestion of the seven inserts have been successful and individualized plasmids of each gene have been obtained.

Subsequently, pairwise ligation of the inserts was performed as previously described. Correct inclusion of the gene pairs into a plasmid with a different antibiotic resistance was observed. Although the results were conclusive in that the digests were performed correctly, the ligation of the two inserts had very low efficiency, resulting in a low number of colonies or even empty plates. Even so, the ligation of the plasmid containing the cellulolytic genes was performed correctly.

Finally, the colorimetric test for the functionality of the psBC1Cel1 plasmid was performed. The congo red test was positive in two of the eight samples tested, showing a hydrolysis halo around the bacterial growth. However, the resulting halo is similar to that observed in the literature, indicating a good degradation efficiency.

Future perspectives

Due to time constraints, it was only possible to verify the assay to determine the hydrolysis capacity of cen and cex cellulases, obtaining cellobiose as a product. For this purpose, the commercial reagent m-methyl cellulose was used, which made it possible to make the culture plates on which the presence of this reaction was revealed with congo red (for more information on the procedure, see the section Verification of Experiments).

Once the correct performance using such a substrate has been determined, future alternatives are proposed to use other cellulose-rich sources, such as plant waste itself, as well as other products such as porous filter paper. The extraction of the cellulose-rich fraction and its subsequent development in the same way will allow the determination of the stereospecificity of this reaction, as well as an optimization of the gene construction for its maximum yield.

However, a more in-depth study is not excluded in which the possibility of the bacteria being able to work directly on plant residues without the need to homogenize and treat them is considered.