Part:BBa_K2958010

Single Chain Insulin Gene Block (with tags for purification)

This single chain native insulin contains an extended RBS (iGEM17_Sydney_Australia), ecotin tag(BBa_K2417010), a 6GGS-6His tag-6GGS linker (BBa_K2958003), TEV tag (BBa_K2958000), human proinsulin sequence coding sequence from iGEM17_Sydney_Australia (BBa_K2417006), and double terminator (B0015).

Description:

Proinsulin is normally made of an A Chain, B Chain, and C chain. Insulin is in its active form after the C chain is cleaved by endopeptidases. Specifically, prohormone convertases (PC1 and PC2). However, with the addition of the GGYLGGGGGGGR linker, the insulin-producing process no longer requires this step. The PI of this single chain insulin is 5.50, which is close in value to native insulin’s PI of 5.40. We designed this single chain insulin with the intent to compare its structure and function to native human insulin in hopes of confirming that the single chain insulin has a similar reaction rate as the wildtype.The proinsulin gene sequence contains 3 different tags for purification--an ecotin tag that is meant to send our Insulin to the periplasm of the cell for proper disulfide bond formation, a 6GGS-6 His tag-6GGS tag that is meant to aid in the first step of our insulin purification via nickel beads, and a TEV tag used for the site-specific cleavage of the his tag. It was important for the ecotin tag to be at the end of the protein in order for the proinsulin disulfide bonds to properly form in the periplasm, so the 6 his tag fell between multiple tags. To ensure the his tag is still functional, we added 2 6GGS spacers based on the iGEM17_Sydney_Australia design to increase flexibility of the his tag and allow for purification.

DIagram of Insulin Gene Block Purification Figure 1. A step by step diagram of the insulin purification process.

Gel Electrophoresis Confirmation of Proinsulin Gene Block

Figure 2. Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is PCR purified LacP+RFP at the correct base pair length (924 bp), lane 3 is PCR purified proinsulin at the correct base pair length (1068 bp), NL 5.50 Single Chain Proinsulin at the correct base pair length (1002 bp), and lane 4 is long lasting single chain insulin at the correct base pair length (1002 bp). This gel electrophoresis result supports that the single chain native insulin gene block with tags for purification was properly PCR'd.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 552
  Illegal BamHI site found at 624
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 589