Team:SDU-Denmark/Tour23

System design

Think big – safe the world!

Original thought

The original design of the system making up the Edible coli, is a K12 MG1655 strain of Escherichia coli:

1. Excreting cellulases for the degradation of cellulose to glucose:
   Cellulose (C₆H₁₀O₅)_n consists of β-1,4 linked D-glucose units. For the Edible coli to gain nutrients from cellulose in the form of glucose units, the β-glucosidic bonds in-between must be broken by hydrolysis. For this degradation, three enzymatic activities are needed by the enzymes, collectively known as cellulases: Endoglucanase, exoglucanase and β-glucosidase. Endoglucanase hydrolyses internal β-1,4 glucosidic bonds in the cellulose fiber, while exoglucanase hydrolyses the external bonds, releasing cellobiose disaccharides. The cellobiose disaccharides are then cleaved by β- glucosidase into two glucose molecules each. Source: Lynd, L.R., et al.: Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews, 2002. Vol. 66:3, p. 506-577. (Link)
   
   Reaction and biobricks needed for the reaction to run (Edinburgh, 2008):
   
   
2. Producing a nutrional, self-designed protein – the OneProt:
   Among others, using the glucose from cellulose degradation as a nutrition source, the Edible coli will be able to produce a high quantity of essential amino acids, incorporated into a self-designed protein.
   
   

   One protein to rule them all!

   The OneProt design:
   The nutritional protein consists of the right ratio between essentials amino acids, and between essential and non-essential amino acids, needed in the daily diet, as recommended by the WHO/ FAO/UNU Expert Consultation. Source: WHO/FAO/UNU Expert Consultation: Protein and Amino Acid Requirements in Human Nutrition. United Nations University, 2002. No. 935, p. 164. (Link) The optimal rates of essential amino acids are shown in table 1.
   
   The protein sequence encodes 480 amino acids in total. The amount of essential amino acids is based on the recommended ratio between essential and non-essential amino acids, which should be 27.7 %. Source: WHO/FAO/UNU Expert Consultation: Protein and Amino Acid Requirements in Human Nutrition. United Nations University, 2002. No. 935, p. 150. (Link) The rates of given essential amino acids are based on the recommendations in table 1, and the rates of given non-essential amino acids are based on how big an amount is used and processed in the body, and whether they can be formed from essential amino acids or not.
   
   In all, the protein comprises 136 essential (28.3 %) and 344 non-essential (71.6 %) amino acids, with the distributions shown in table 2 and table 3. To make sure the design would not contain any harmful protein structures or misfold, the sequence has been shuffled, codon optimized for E. coli, K12 MG1655, checked for restriction sites and its structure has been predicted, using Phyre²
   
   The expression of this coding sequence (basic part: BBa_K1475001), and thus the synthesis of nutritional protein will be regulated by the repressible promoter, pTet (BBa_R0040):
   Expression of OneProt regulated by repressible Tet promoter.
   
   

   The activity of pTet regulated by the repressor protein, TetR (BBa_C0040), should be tested with and without LVA-tag by regulating the expression of GFP:

   Expression of GFP regulated by repressible Tet promoter: TetR(+LVA) vs. TetR(no LVA).

   GFP controlled by TetR(+LVA)-pTet (BBa_K1475006 ) GFP controlled by TetR(no LVA)-pTet (BBa_K1475005)