Team:HAFS/Applied Design

Design

Overview

Minicell

Figure 1. The mechanism by which minCDE gene prevents asymmetric cell division

The minCDE gene, or the genetic locus made up of minC, minD, and minE is responsible of preventing asymmetric cell division at the poles. More specifically, MinD, when bound to ATP, anchors at the cell poles (and cell poles, only) and polymerises to form a cluster. These clusters activate MinC by binding and inhibits FtsZ polymerisation at the poles. As a result, the division is initiated at the centre of the cell and not at the poles. [1,2] Hence, when minCDE locus is deleted, FtsZ can polymerise not only at the mid-cell but also at the poles. If this happens, a small, spherical mini cell without chromosome formulates. [3]

There are two important characteristics of mini cells. First, because is does not have any bacterial chromosome, the cells do not divide anymore. Secondly, unlike bacterial chromosome, plasmids can segregate into the mini cells. Hence, the cells will synthesise protein encoded by the plasmid DNA. All things considered, the non-propating mini cells are much more easily containable than normal bacteria, making it appropriate for the delivery of peptide drug.

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Insulin

   Winsulin is an open source single chain insulin developed by Team Sydney Australia (iGEM17). We decided to use Winsulin for few reasons. The key difference between Winsulin and Human Insulin is that the Winsulin has 12 amino acid linker protein between A chain and B chain. The 12 amino acid linker protein allows for the two chains to flexibly form disulphide bond for accuracy. We chose to use Winsulin for three reasons. First, the obvious advantage was that we d not infringe any patent when submitting the part. But secondly, because Winsulin is a single chain insulin that has higher stability and activity than human proinsulin that requires further processing. Lastly, Winsulin is a rapid acting insulin, which is suitable for bolus insulin, that which our mini cells should deliver.

   Cell penetrating peptides facilitate cellular intake of its cargo, which in this case is the Winsulin. Normally, Insulin cannot penetrate the epithelial cells of intestine, which makes it inappropriate for oral intake. However, when associated with penetratin, the cell penetrating peptide that we used in this design, does not only facilitate the absorption of insulin through intestine but also increases the half life of insulin more than eight times in vivo. [1]

   We designed three different construct using three constitutive promoters: J23100, J23106, J23116. These three promoters have different promoter strength, J23100 being the strongest and J23116 being the weakest. Dose control is an important parameter during insulin therapy because overdose of insulin can cause hypoglycaemia and weight gain which can be detrimental to diabetic patients. Hence, we thought it was necessary to regulate the amount of insulin secreted by the strength of promoter.

   Lastly, we designed our construct to contain flag tag in the end so that it can be easily detected during immunoglobin assay such as western blot.

Aoki, Yoshinobu, et al. “Region-Dependent Role of the Mucous/Glycocalyx Layers in Insulin Permeation Across Rat Small Intestinal Membrane.” Pharmaceutical Research, vol. 22, no. 11, 2005, pp. 1854–1862., doi:10.1007/s11095-005-6137-z.

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Lysis based secretion

   The last part of our minicell-based bolus insulin delivery is lysis based secretion, in which the cells lyse in the intestine to break open and secrete the Winsulin produced within the cell.

   Here, the bolus insulin should decrease the blood glucose level right after food intake. We decided to use the increase in concentration of bile salt during digestion as a signal that induces the lysis gene. Bile, bade up of bile salts and phospholipid, is produced in the liver and stored in gallbladder before being secreted. When fatty acids enter the duodenum, hormone cholecystokinin (CCK) is secreted, stimulating the gall bladder to secrete bile into the duodenum. Hence, the high concentration of bile salt can be an apt signal to food intake. [1]

   Furthermore, this construct only contains T4 endolysin and not T4 endonuclease, considering the mini cells do not have any chromosomal DNA.

[1] Hofmann, Alan F. “Overview of Bile Secretion.” Comprehensive Physiology, 2011, doi:10.1002/cphy.cp060328.

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Team: AQA_Unesp