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Team:UCL London/Medicine/ImpactAndSynergy
From 2011.igem.org
Improvements we're making to plasmid technology
Not only does the gene coding for the desired antigen need to be expressed by E.coli but according to the Food and Drug Administration (FDA), a prescribed amount of 80% total supercoiling for genes that need to be expressed for DNA vaccines. Greater than 80% of supercoiled pDNA must be obtained for other plasmid DNA technologies. Supercoiled pDNA is usually considered to be more effective at transfecting cells than open circular and linear variants, and therefore mount a better immune response. Using our E.coili manufacturing toolkit we can go about finding a more efficient, cost effective method for meeting these requirements.
Our Supercoiliology sub-project focuses on expressing gyrase A and B subunits in an E.coli cell using a promoter not available in wild type DNA gyrase, which doesn´t trigger the negative feedback mechanism limiting the expression of gyrase in the cell. This leads to an increase in the amount of gyrase expression which in effect, allows a greater amount of supercoiled pDNA to be formed. Having a greater concentration of pDNA in the upstream product produces a higher standard of purity, facilitating subsequent downstream processing steps provides a final product which is better enabled to meet the 99.9% purity standard set by medical regulatory authorities. Having a product of high purity is necessary especially in the case of vaccines being administered to humans. This is particularly relevant to vaccination schedules where repeated dosages must be given. If there´s a high enough concentration of contaminants in the vaccine, these contaminants will be compounded in the body overtime, this may have dire consequences.
Magneto- Sites creates greater specificity of target plasmids for gyrase through the addition of binding sites to provide preferential supercoiling of our pDNA vaccine vector rather than genomic DNA which would otherwise interfere with cell growth. This increased specificity for targeted plasmids will produce a greater yield of plasmids that have a higher superhelical density, indicating higher quality of supercoiling while also increasing the efficiency of this process. Increased supercoiling allows enhanced transfection of the desired gene insert into the cell due to the greater compactness of the gene of interest. Increased compactness of genes is also beneficial for future applications when it comes to delivering multiple vaccinations at once since these huge gene sequences can all be compressed and input into the cell at once. Tighter supercoiling provides greater robustness of DNA causing it to be less susceptible to damage. Plasmids are fairly stable at room temperature, again in contrast to a number of live vaccines whose storage and global delivery are complicated by the need to keep the vaccines cold, so it can be delivered to patients in lesser developed countries that don´t have such facilities.
Extractery provides the means to increase product purity by removing DNA that doesn´t meet a threshold standard established by the FDA. Also, it frees the remaining supercoiled plasmid into the cell lysate enabling a faster, more robust downstream purification process which should cut down production costs greatly, allowing cheaper vaccine production. This sidesteps a major manufacturing bottleneck, saving us time.
Stresslights 2.0 and Supercoilometer are tools we´ve designed in order to monitor the growth condition of the cells containing the desired insert. It provides us with a means to ensure that each batch meets regulatory standards and allows consistency between batches enabling a much more standardized product overall.
