Team:Hong Kong HKUST/Project/module4

Overview

Our artificial futile cycle design is based almost entirely on the tested findings by Dean et al, who demonstrated that by introducing the artificial glyoxylate shunt in mouse liver cells, fatty acid uptake would increase and the mice would acquire resistance against obesity when fed with fatty diet. (IN TEXT CITATION) In essence, we are reproducing their work from scratch but through the use of standard BioBricks. [Figure from Dean et al’s paper] In Dean et al.’s work, the glyoxylate shut was achieved by the expression of two key enzymes from the bacterial glyoxylate cycle, isocitrate lyase (AceA) and malate synthase (AceB). When the two enzymes enter mitochondria in liver cells, isocitrate lyase will convert a proportion of isocitrate into glyoxylate, which will then be converted by malate synthase into malate. This process would bypass the pathway through alpha-ketoglutarate, and therefore, bypassing the ATPs and reducing equivalent generating steps. (IN TEXT CITATION) To reproduce this masterpiece, we would first need to convert every single part into BioBricks: we cloned out the glyoxylate enzymes genes aceA and aceB from E. coli and assembled them with mitochondrial leader sequence (MLS). The two translation units were then assembled downstream of mammalian constitutive CMV Promoter and EF-1alpha Promoter respectively. Lastly, the hGH polyA signal sequence was added to serve as terminator of the construct. These constructs, when put together, should return the original constitutive glyoxylate shut. Yet, in addition to the constitutive system, we are assembling a fatty acid inducible construct that allows tunable gene expression according to the concentration of fatty acid around. We decided to introduce this inducible system to prevent fatty acid deficiency when the concentration of fatty acid in body is low, hopefully overcoming the foreseeable shortcomings of the original constitutive shut. Lastly, the inducible and constitutive system will be compared in terms of fatty acid uptake rate in a range concentration of fatty acid and their performances shall be evaluated. Reference: Dean ……..