Part:BBa_K2958012
Fast Acting Single Chain Insulin Analog Gene Block (with tags for purification)
This single chain proinsulin analog contains an extended RBS (iGEM17_Sydney_Australia), Ecotin tag (BBa_K2958015), a 6GGS-6 his-6GGS linker (BBa_2958003), TEV, fast acting single chain proinsulin sequence (BBa_K2958008), and double terminator (B0015).
Description:
This Fast Acting Single Chain Insulin Analog was designed to have a PI of 5.50, the same PI as Insulin Lispro. Modifications include the single chain linker (BBa_K2958004) and a lis-pro amino acid switch at the B28 and B29 position on the B-Chain, which is the same modification as Lispro (unpatented as of 2019). We designed this single chain fast acting insulin with the intent to compare its structure and function to native human insulin in hopes of confirming that the fast acting insulin has a quicker reaction rate than the wild type. 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.
Design Consideration: We experienced difficultly with producing a pure Fast acting Insulin gene block pcr product due to the difficulty in the primers not properly binding to the gene block. To improve this, we incorporated the overlaps into the gene sequence. Although this slightly improved the results, we were unable to produce a successful fast acting insulin gene block. (See figure 2.)
Figure 2 (A) Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is 1 kb ladder ladder, lane 2 is our unmodified fast acting insulin gene block (without overlaps for improved primer binding during pcr). The expected base pair length for this fast acting insulin gene block was 1002 bp. As seen in the gel results, the pcr product of the fast acting insulin gene block resulted in a smear. Typically, this is due to high DNA template concentration prior to the PCR reaction. However, the gel shown above is representative of all of our attempts. Note: the annealing temperature for these reactions were 57C, as calculated by the NEB TM Calculator (B) Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is fast acting insulin with overlaps with an annealing temperature of 59C, lane 3 is fast acting insulin with overlaps with an annealing temperature of 61C, and lane 4 is fast acting insulin with overlaps with an annealing temperature of 63C. The annealing temperature for these reactions were increased to decrease the likelihood of smearing by allowing the primers to bind to the DNA. The expected base pair length for this gene block is 1032 base pairs. It appears that adding the overlaps to the construct have helped the pcr reaction of this gene block according to the result in lane 4.
Fast Acting Insulin Sequence: TTTTGTTTAACTTTAAGAAGGAGAAAGCTTATGTACTAGATGAAGACGATCTTGCCAGCGGTGTTATTCGCCGCGTTCGC GACAACATCTGCCTGGGCCGCAGAATCAGTTCAGCCGTTGGAAAAGATTGCGCCCTATCCGCAGGCTGAAAAAGGTATGA AGCGTCAAGTCATCCAGTTGACTCCACAGGAAGATGAATCTACCCTTAAAGTAGAATTACTTATCGGTCAGACTTTGGAG GTAGACTGCAACTTGCACCGTTTGGGGGGAAAGTTGGAGAACAAAACTTTAGAAGGATGGGGTTATGATTATTATGTATT CGACAAAGTTAGTAGCCCTGTCTCCACTATGATGGCATGTCCTGATGGCAAAAAGGAAAAGAAGTTCGTGACAGCATACC TTGGTGACGCTGGAATGTTGCGTTATAACTCAAAGTTGCCGATTGTCGTTTACACGCCAGATAACGTTGACGTCAAGTAT CGCGTATGGAAAGCCGAGGAAAAGATTGATAATGCGGTAGTACGTAAATTGGGGGGTTCGGGCGGCTCAGGTGGCTCAGG GGGGTCAGGCGGGAGCGGGGGGTCACACCATCATCATCATCATGGAGGCTCAGGAGGGAGTGGGGGTTCGGGAGGCTCGG GTGGCAGCGGAGGTTCCGAGAATTTATATTTTCAGAGTTTTGTGAACCAACACTTATGTGGTTCTCATCTGGTAGAGGCG CTTTATTTAGTGTGCGGGGAGCGTGGATTTTTTTATACGAAACCGACCGGAGGCTATCTGCCCGGTGGAGGGGACGTTGG TCGTGGAATCGTAGAGCAATGTTGCACATCCATCTGTTCGCTTTACCAGCTTGAAAATTACTGTAACTGATAACCAGGCA TCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGA GTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
Fast Acting Insulin with Overlaps Sequence: TGTAGATCGCTTTTGTTTAACTTTAAGAAGGAGAAAGCTTATGTACTAGATGAAGACCATTTTGCCGGCGGTCTTGTTTG CAGCATTTGCAACTACTTCAGCTTGGGCTGCAGAGTCAGTGCAACCTCTGGAGAAAATCGCTCCTTACCCGCAGGCAGAG AAGGGCATGAAACGCCAAGTTATCCAATTGACACCACAAGAGGACGAATCTACCTTGAAAGTAGAACTGCTGATTGGTCA AACGCTGGAGGTTGATTGCAATCTTCACCGCTTGGGTGGGAAGTTAGAAAATAAAACGTTGGAAGGCTGGGGCTACGACT ATTACGTCTTTGATAAGGTATCGTCGCCCGTATCGACTATGATGGCTTGCCCCGATGGGAAGAAGGAGAAGAAATTCGTG ACAGCATATTTGGGAGATGCCGGCATGTTACGTTACAATTCAAAGCTTCCAATTGTTGTCTACACTCCGGACAATGTGGA CGTGAAGTATCGTGTCTGGAAGGCTGAGGAGAAAATTGACAACGCCGTTGTCCGCAAACTGGGTGGCTCTGGCGGATCCG GTGGCTCGGGTGGTTCGGGTGGATCAGGAGGTTCTCATCATCATCACCACCACGGAGGGTCTGGAGGATCAGGAGGATCA GGGGGTAGCGGGGGTAGCGGGGGCTCAGAGAACTTGTACTTTCAGTCTTTCGTAAATCAGCACCTTTGTGGCTCCCACCT TGTGGAGGCACTTTACTTGGTATGTGGCGAACGTGGCTTCTTTTATACAAAACCAACCGGAGGGTATCTTCCCGGTGGAG GTGACGTGGGACGTGGGATCGTGGAGCAATGTTGTACGAGCATTTGTTCGTTATATCAATTAGAAAATTATTGTAACTGA TAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCT CTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCA
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 56
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 56
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 56
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 56
- 1000COMPATIBLE WITH RFC[1000]
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