Part:BBa_K2958009

Native Proinsulin Gene Block (with Tags for purification)

This native Proinsulin contains an extended RBS (iGEM17_Sydney_Australia), ecotin tag(BBa_K2958015), 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:

This proinsulin sequence is the negative control for our experimental circuits.This gene block will allow us to compare the structure and function of our single chain insulins (single chain native proinsulin, fast acting single chain insulin, and long lasting insulin) to wildtype insulin. The proinsulin gene sequence is based on the 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 (not used for this project; 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 Proinsulin gene block with tags for purification was properly PCR'd.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 56
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 56
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 56
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 56
  
• 1000
  COMPATIBLE WITH RFC[1000]