Part:BBa_K2958014
LacP with RFP + Long Lasting Insulin gene Block
This composite part contains RFP regulated by a Lac promoter (BBa_K2958002), and long lasting insulin gene block (BBa_K2958007) that has been modified in the A Chain in position 21 (from an Asparagine to an Alanine) to reflect a similar mutation to the patented Long Lasting Insulin, Glargine. Additionally, a GGYLGGGGGGGR linker (BBa_K2958005) has been added in order to get the PI at 6.46, which is close to the PI of the long lasting insulin Glargine (6.7)
Description
The LacP + RFP portion of this composite part functions as the reporter gene used to confirm the expression of a circuit. RFP is easily visible, and the cells will glow red if it is properly expressed. This circuit is regulated by the Lac Promoter. The promoter will act as a constitutive promoter because there is no Lac repressor in this construct. However, the promoter still contains the binding site for the lac repressor native to E. coli, this construct can be treated with IPTG as a precaution to ensure expression.
The long lasting insulin gene block has been modified in the A Chain in position 21 (from an Asparagine to an Alanine) to reflect a similar mutation to the patented Long Lasting Insulin, Glargine. Additionally, a GGYLGGGGGGGR linker (BBa_K2958005) has been added in order to get the PI at 6.46, which is close to the PI of the long lasting insulin Glargine (6.7) We designed this single chain long lasting insulin with the intent to compare its structure and function to native human insulin in hopes of confirming that the long lasting insulin analog has a slower reaction rate than wild type insulin. The long lasting insulin 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.
Figure 3. (A) 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 RFP gene block and the long lasting insulin gene block with tags for purification was properly PCR'd. (B) Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is PCR purified psB1C3 plasmid at the expected base pairs (2070 bp). This psB1C3 plasmid was used as the plasmid backbone for this LacP with RFP + Proinsulin gene block with tags for purification composite part.
Figure 4. Transformation of RFP + Long Lasting insulin Gibson Assembly into DH5-Alpha (E.coli) cells exhibits colony growth.
Figure 5.Colony PCR of RFP+Proinsulin construct. This colony PCR isolates our construct form the plasmid, and is used as a confirmation test of our gibson assembly. The RFP + Long lasting Insulin gene blocks result in a complete construct that is 1926 base pairs long. (RFP is 924 bp, and Long Lasting Insulin is 1002 bp; 924 bp + 1002 BP = 1992 bp). These colony screening results indicate a construct at ~2000 bp, as the band is beneath the 2 kb band. From these results, we conclude that the Gibson Assembly was successful.
Figure 6. EcoRI and PstI Restriction Digest of RFP + Long Lasting Insulin (left) versus Predicted Results of Proinsulin Restriction Digest. According to the virtual restriction digest conducted on serial cloner, a restriction digest of the RFP + Proinsulin construct will result in 2 fragments: one 2,033 bp fragment and one 1,963 bp fragment. The two fragments at about 2,000 base pairs in length will appear as one band due to their similarity in length. Based on the results of the restriction digest, our bands appear to be at the expected fragment length. Through this digest, we were able to locate an illegal pstI site in our proinsulin fragment. However, analysis on serial cloner confirms that this pstI site does not affect the protein sequence, and this composite part is still RFC[1000]/Type IIS compatible. According to this data, the gibson assembly reaction of the RFP + Proinsulin gene block composite part was successful.
Figure 7.EcoRI Digest of RFP + Long Lasting Insulin (left) versus Predicted Results of Proinsulin Restriction Digest. This digest was performed in order to confirm the entire construct including the psB1C3 plasmid is present to further confirm successful Gibson Assembly, particularly because the two fragments generated from the Digest with Eco-RI and PstI are similar in size at 2,033 base pairs and 1,963 base pairs. According to the virtual restriction digest conducted on serial cloner, a restriction digest of the RFP + Proinsulin construct will result in 1 fragment at 3,996 base pairs.Through this digest, we were able to locate an illegal pstI site in our proinsulin fragment. According to this data, the gibson assembly reaction of the RFP + Proinsulin gene block composite part was successful.
Figure 8.Protein gel of the RFP + Long Lasting Insulin construct, courtesy of iGEM Team_Moscow 2019. According to this protein gel, our construct appears to have worked as expected, as seen by the bands at the expected proinsulin fusion construct band and expected RFP band in the (+) lysate of the protein gel. (indicated by the red arrow). The expected molecular mass of the long lasting insulin is 29.08 kda, and the expected molecular mass of the RFP is 25.38 kda.
Figure 1. Figure 9. (A) Protein model of native human insulin (active form) produced on Swiss Model software. Orange portion is A chain of insulin, and green portion is B chain of insulin. (B) Protein model of long lasting single chain insulin with GGYLGGGGGGGR linker (BBa_K295005) produced on Swiss Model software. Orange portion is A chain of insulin, green portion is B chain, and red portion is the linker.
Figure 10. Protein model of Native human insulin (orange portion = A chain of native insulin, green portion = B chain of insulin) superimposed on protein model of single chain long lasting insulin (pink portion = A chain of long lasting single chain insulin, blue portion= B chain of long lasting single chain insulin, red portion = GGYLGGGGGGGR linker) produced on Swiss Model Software. Based on these superimposed models alone, it appears that the addition of the linker does not disrupt the structure of the A and B chains. However, the side chains of these insulin molecules require more extensive analysis.
Figure 11. Figure 3. Protein model demonstrating interaction energy (DG) between the single chain native insulin molecule and the ectodomain of the insulin receptor (domain outside of cell membrane). The estimated DG is 1.84606 kcal/mol. In comparison to the estimated DM of our single chain insulin (BBa_K2958006), which was 2.04839 kcal/mol, indicating that the mutations in the long lasting insulin analog decreased the interaction energy by 0.20233 kcal/mol. This model was produced with FoldX software, courtesy of iGEM Team_Moscow 2019.
Figure 12. Protein analysis of the stability of Long Lasting Insulin, conducted by iGEM Team_Moscow with a OPLS AA force field (A.) Analysis of RMSD to check stability of Long Lasting Insulin Analog. The RDSM exhibits a plateau, indicating that this analog is stable. (B) Analysis of the radius of gyration of the long lasting insulin analog to measure compactness. The value of rg is relatively steady, indicating stable protein folding.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1492
Illegal BamHI site found at 1564 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1529
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