Team:Aix-Marseille/Protocols
Although algae is a potential bioethanol feedstock, the production from untreated macroalgal wastes is inefficient. It’s the most cost and difficult step of ethanol production.The objectives of an effective pre-treatment are to obtain a concentrated algae biomass and facilitate the next-step biological degradation. It’s important to choose the optimal process for preventing lost or degradation of obtained sugars, limiting the toxic materials which inhibit the ethanol production, reducing energy requirement for process and minimizing the production cost allowing to obtain the best yield of ethanol at the end.
Algae pre-treatment : Washing Drying and Grinding
Algae samples were washed with tap water to remove impurities like sand, shellfish, and other materials. Then algae are dried in an oven at 70 °C for 24 h. Dried samples were ground to improve the pre-treatment efficiency, and stored in a clean air-tight container.
After that, the slurry biomass would be added in the medium with E.coli W3110 ΔrhaT/rhaB strain to enhance rhamnose extraction from polysaccharides and then would be metabolized during the fermentation step which will be developed next year.
Strain transformation and enzymes production protocols:
To the beginning, one by one, those plasmids would be integrated and amplified in the DH5alpha strain. Because DH5alpha hasn’t the gene RecA, This strain can’t do homologous recombination. Plasmids would be extracted and purified using miniprep protocols in order to be integrated into a E.Coli W3110 ΔrhaT/rhaB by electroporation. RhaT is a rhamnose transporter and RhaB the first protein of rhamnose metabolism. We would have a strain which is not able to metabolize rhamnose because our aim is to quantify the amount of rhamnose produced by the strain.
Once plasmid is integrated in the strain, we will induce the gene expression by adding IPTG. Plac promoter IPTG-dependant, is activated and begins transcription of the inducer genes fused with hlyA transport signal. The desired protein is secreted through the membrane via T1SS. Finally, thanks to the tag sequence, we would purify the 4 proteins with an affinity column. After these production steps, we wish to identify our proteins secretion and by products of ulvan degradation thanks to HPLC-MS.
Once the activity of the 4 proteins would be measured, we could then build our final design. E.coli W3110 ΔrhaT/rhaB will be transformed with two plasmids by electroporation due to the size of the plasmids: 1 with 3 genes and 1 with the gene coding for sulfatase which is much larger. Overproduction the 4 enzymes passing through the same secretion system, risks cluttering it and preventing optimal secretion in the medium. In this case, we have thought to overexpress the genes coding for the T1SS. Moreover, the TEV protease will be overproduced in the culture medium to cleave HlyA signal sequence, allowing maturation proteins and to activate their catalytic activity. Removal of the secretion domain would be confirmed using SDS-PAGE and Western-Blot.
Finally, we would identify the enzymatic activities by examining the products of ulvan degradation using HPLC-MS.
Rhamnose analysis :
An E.coli W3110 strain would be transformed with chromosomal rhaS (Rhamnose sensor) and a plasmid containing a biosensor (GFP) under control of a promoter PrhaBAD, thus rhamnose-dependent. The same transformation process than explained above could be used.
For each step, rhamnose concentration would be determined thanks to our biosensor system. A sample of the reaction medium will be transfered into the medium containing E.coli biosensor strain. When they have some rhamnose to the media, the chromosomic rhaS will express the transcriptional activator of the PrhaBAD promoter. The strains will produce GFP fluorophore. GFP emission will be measured thanks to an Fluorescence microplate titer. and will allow us to know the rhamnose concentration !
This system is very useful in comparison with other inducible promoters because it allows a high expression but is undetectable without the inducer (L-rhamnose). It has also the advantages to be dose-dependent so not with a mechanism of « all or nothing », and it’s non-toxic too. However, during our research to do an efficient biosensor for the rhamnose, to assess if our strains produce these monosaccharides well, we have found 3 articles which gives us the possibility to increase the efficiency of this iGEM biobrick and give a useful contribution.