Team:Sydney Australia/Improve

Old Parts

Parts BBa_M39904 and BBa_M1877 were pre-existing registry parts described as ‘Human Insulin cDNA’ and ‘human insulin’ respectively.

What we Improved

Part BBa_M39904 (Figure 1) consisted of 345 base pairs and was submitted as a basic part, and showed no differentiation between A and B chains, or if there was a C-peptide present.

Part BBa_M1877 (Figure 2) consisted of only 12bp and so was clearly unfinished.

Figure 1: BBa_M39904 sequence as displayed in registry. The part is not compatible with many biobricks, and has no differentiation between A and B peptides.

Figure 2: BBa_M1877 sequence as displayed in registry. As human insulin is clearly more than 12bp this sequence was deemed incomplete.

Our Improved Parts

We submitted BBa_K2417000, BBa_K2417001 and BBa_K2417006 as improvements upon these (Figure 3).

Figure 3: BBa_K2417000 – Ecotin-Proinsulin, BBa_K2417001 – Cytoplasmic-Proinsulin, BBa_K241006 - Proinsulin – are all improvement parts because they are much more clearly characterized and contains features for more efficient production and purification.

Summary

Previous Part	Characteristic	How did we improve this?	Which parts did we submit with this improvement?
BBa_M399	Basic part	We submitted composite parts that clearly distinguish between A and B chains, and the presence of a C peptide. The use of a composite part submission system allows users to access the basic parts they consist of for more detailed information.	BBa_K2417000 BBa_K2417001
	Lacking complete sequence	Our parts are sequence verified, confirmed as complete and are based off NCBI sequences of human proinsulin.	BBa_K2417000 BBa_K2417001 BBa_K2417006
	Lacking ribosomal binding site	Our parts contain an efficient extended ribosomal binding site (part BBa_K2417009) which aids successful expression.	BBa_K2417000 BBa_K2417001 BBa_K2417006
	Lack of purification tags	We added an N-terminal His-tag (BBa_K2417008) to all of our proinsulin constructs to aid ease of purification. A His-tag allows purification using an affinity chromatography column.	BBa_K2417000 BBa_K2417001 BBa_K2417006
	No secretion tags	For some of our constructs we added tags to induce secretion of the protein to different parts of the cell. The addition of Ecotin to the N-terminus of proinsulin has been shown to induce its transport into the periplasm of E. coli, an oxidative environment favourable for the folding of disulphide bonds.	BBa_K2417000
BBa_M39904	Biobrick incompatible	The construct entered into the database is only compatible with a single biobrick. All of our parts are compatible with all biobrick constructs.	BBa_K2417000 BBa_K2417001 BBa_K2417006