Difference between revisions of "Part:BBa K2408020"

Revision as of 06:42, 16 October 2017

ADH1_KP6_proToxin

KP6 protoxin for inhibiting wine spoilage by Brettanomyces

Alcohol Dehydrogenase 1 (ADH1) is a strong constitutive promoter isolated from Saccharomyces cerevisiae. The toxin KP6 binds to the outer membrane of the Brett using receptors and damages the protective membrane of the yeast and thus causes the death of the Brett cell. This toxin does not affect the wine yeast (s.cerevisia) so there is no problem using it. It functions maximally between temperatures 5-20 degrees Celsius and PH ranging from 2.5-5 and the results on the subject found that it affects many types of Brett and therefore we decided to use it. we also added the α-mating factor secretion signal and a histidine tag.

Design: Objective: Toxin gene cloning to preserve S. cerevisiae to give it the ability to inhibit harmful yeast in the wine industry - Brettanomyces . The gene to KP6 is taken from yeast of U.maydis. KP6 could inhibit Brettanomyces but did not harm S. cerevisiae. Difficulties: KP6 is in a state of protoxin- before it is fissioned by protease, and the fission of the source (U.maydis) occurs and produces two subunits of the toxin (alpha and beta), in which S. cerevisiae may not be cleaved identical to the original, Expressing the gene in the state of protoxin and in subunit mode when they are expressed separately by adding a Kozak sequence between them. This is why we added two parts ( BBa_K2408021, BBa_K2408022 ) that contain both subunits (alpha and beta) of the toxin separately. In addition, we want the toxin to be both constitutive and inducible to ethanol or sugar so that the expression of toxin can be controlled at the desired time of fermentation. Baccalaureate test: Brett's growth delay test at the level of S. cerevisiae transgenic

· Santos, A., Navascués, E., Bravo, E., & Marquina, D. (2011). Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. International journal of food microbiology, 145(1), 147-154

· Tao, J. I. A. N. S. H. I., Ginsberg, I. D. I. T., Banerjee, N. A. N. D. I. T. T. A., Held, W., Koltin, Y. I. G. A. L., & Bruenn, J. A. (1990). Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Molecular and cellular biology, 10(4), 1373-1381.‏

. Lin-Cereghino, G. P., Stark, C. M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., ... & Huang, A. D. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene, 519(2), 311-317.‏

Sequence and Features