Adaptive Modeling Software

The competitive relationship between Ex-4 and GLP-1 is unique in that both proteins cause the same downstream effect: insulin secretion from pancreatic beta cell islets [1]. Extensive research went into selecting an appropriate equation to provide a stable foundation while building our competitive binding model. To assist future iGEM teams studying similar competitive binding relationships, we programmed our model to be adaptive and open source. Anyone can download our code and generate custom graphs by changing the input values to match their protein dissociation constants, initial concentrations, and binding formulas. Features such as panel formatting, EC50 plotting, and legend configuration are handled automatically. To learn more about our computational models, see the modeling page.


Parts Collection

Our project required the development of twenty-five new basic and composite genetic parts for the expression of Ex-4 in both S. cerevisiae and E. coli. Future iGEM teams interested in increasing the oral bioavailability of Exendin-4 as a T2D therapeutic can use and build upon the parts we created and submitted. The basic parts can be used in more general applications for teams looking to assemble plasmids for recombinant protein production. The parts submitted for S. cerevisiae use the Modular Cloning (MoClo) library, allowing those interested in assembling a plasmid via MoClo to use parts we designed and verified.

Our protein transcribing gene inserts generally include a 6xHistidine-Tag, glycine-serine amino acid linker, protease cut site, and codon optimized Ex-4 sequence. In E. coli, the protein was expressed by the pET28 plasmid whereas for S. cerevisiae we inserted our gene directly into the genome at either the LEU2 site or the TRP1 site as two separate testing points. Additionally, we constructed a gene insert for pET28 to express the truncated GLP-1 R binding domain to test the functionality of our Ex-4 protein. The parts we designed for this project are listed in the tables below:

Basic Parts

Part Name Part Number Part Type Description Designer Length (bp)
pET28:GFP BBa_K4190000 Plasmid Backbone Vector for gene insertion and subsequent protein expression Shis, DL & Bennett, MR. 5993
Shine-Dalgarno Sequence BBa_K4190001 RBS Ribosome binding sequence to ensure genetic code is read in frame Shis, DL & Bennett, MR. 23
6xHis-Tag (E. coli) BBa_K4190002 Tag Histidine tag attached to Ex-4 to conduct immobilized metal affinity chromatography. Codon optimized for E. coli Zoe Petroff 18
6xHis-Tag (S. cerevisiae) BBa_K4190003 Tag Histidine tag attached to Ex-4 to conduct immobilized metal affinity chromatography. Codon optimized for S. cerevisiae. Neo Peng 18
10aa GS Linker (E. coli) BBa_K4190004 Protein Domain Links His-tag to Ex-4 protein. Codon optimized for E. coli Zoe Petroff 30
10aa GS Linker (S. cerevisiae) BBa_K4190005 Protein Domain Links His-tag to Ex-4 protein. Codon optimized for S. cerevisiae Zoe Petroff 30
Enterokinase BBa_K4190006 Other Cut site for eventual removal of His-tag form Ex-4 Zoe Petroff 15
Ex-4 Sequence Codon Optimized for E. coli BBa_K4190007 Coding Region Protein coding region for expressing Ex-4 in E. coli Zoe Petroff 117
GGA Flagged Backbone Primer Forward BBa_K4190008 Primer Flagged primer to add BsaI site to pET28 for GGA Zoe Petroff 47
GGA Flagged Backbone Primer Reverse BBa_K4190009 Primer Flagged primer to add BsaI site to pET28 for GGA Zoe Petroff 43
GAL1 Promoter Plasmid BBa_K4190010 Plasmid MoClo part containing GAL1 promoter for GGA Lee et al. 2204
PGK Terminator Plasmid BBa_K4190011 Plasmid MoClo part containing PGK terminator for GGA Lee et al. 1900
mg-Int-leu2-kan_1420 Backbone BBa_K4190012 Plasmid Backbone Vector for gene insertion and subsequent protein expression Kamakaka, R 5609
mg-int-trp1-hyg_1432-4a Backbone BBa_K4190013 Plasmid Backbone Vector for gene insertion and subsequent protein expression Kamakaka, R 5412
Ex-4 Sequence Codon Optimized for S. cerevisiae BBa_K4190014 Coding Region Protein coding region for expressing Ex-4 in S. cerevisiae Zoe Petroff, Neo Peng 117
EBP10 Half-life Extender BBa_K4190015 Coding Region Extends half life of Ex-4 in vivo Jung, SJ et al. 150
TEV Cut Site BBa_K4190016 Other Cut site for eventual removal of His-tag Zoe Petroff 18
GLP1-R Gene Insert Optimized for E. coli BBa_K4190017 Coding Region Protein coding region for expressing GLP-1R in E. coli Zoe Petroff 365

Composite Parts

Part Name Part Number Part Type Description Basic Parts included Designer Length (bp)
Ex-4 Gene Insert for E. coli BBa_K4190018 Composite His-tagged Exendin-4 coding region to be inserted into a plasmid via GGA and expressed in E. coli
1. Kozak Sequence
2. 6xHis-Tag
3. 10aa GS Linker
4. Enterokinase 
   Cut site
5. Ex-4 sequence 
   codon 
   optimized 
   for E. coli
6. BsaI site
Zoe Petroff 250
Ex-4 Gene Insert for S. cerevisiae BBa_K4190019 Composite His-tagged Exendin-4 coding region to be inserted into a plasmid via GGA and expressed in S. cerevisiae
1. 6xHis-Tag
2. 10aa GS Linker
3. Enterokinase 
   cut site
4. Ex-4 sequence 
   codon 
   optimized 
   for 
   S. cerevisiae
5. EBP10 half 
   life 
   extender
6. BsaI site
Zoe Petroff, Neo Peng 369
GLP-1R Gene Insert in E. coli BBa_K4190020 Composite His-tagged GLP-1R coding region to be inserted into a plasmid via GGA and expressed in E. coli. Expresses only surface binding site.
1. Kozak Sequence
2. 6xHis-Tag
3. 10aa GS Linker
4. TEV Cut site
5. GLP-1R 
   sequence 
   codon 
   optimized 
   for E. coli
6. BsaI site
Zoe Petroff 505
Complete Reconstructed Plasmid (pET28:Ex4) BBa_K4190002 Plasmid His-tagged Exendin-4 expression system in E. coli
1. Kozak Sequence
2. 6xHis-Tag
3. 10aa GS Linker
4. Enterokinase 
   Cut site
5. Ex-4 sequence 
   codon 
   optimized 
   for E. coli
   pET28:GFP
Zoe Petroff 5395
Complete Reconstructed Plasmid (mg-Int-leu2-kan_1420:Ex4) BBa_K4190002 Plasmid His-tagged Exendin-4 expression system with integration at S. cerevisiae Leucine homology sites within the genome
1. 6xHis-Tag
2. 10aa GS Linker
3. Enterokinase 
   cut site
4. Ex-4 sequence 
   codon 
   optimized 
   for 
   S. cerevisiae
5. EBP10 half 
   life extender
6. GAL1 Promoter
7. PGK Terminator
   Mg-Int-leu2
   -kan_1420 
   backbone
Zoe Petroff, Neo Peng 5699
Complete Reconstructed Plasmid (mg-int-trp1-hyg_1432-4a:Ex4) BBa_K4190023 Plasmid His-tagged Exendin-4 expression system with integration at S. cerevisiae tryptophan homology sites within the genome
1. 6xHis-Tag
2. 10aa GS Linker
3. Enterokinase 
   cut site
4. Ex-4 sequence 
   codon 
   optimized 
   for 
   S. cerevisiae
5. EBP10 half 
   life extender
6. GAL1 Promoter
7. PGK Terminatior
   Mg-int-trp1
   -hyg_1432-4a 
   backbone
Zoe Petroff, Neo Peng 5502
Complete Reconstructed Plasmid (pET28:GLP1R) BBa_K4190024 Plasmid His-tagged GLP-1R binding site expression system in E. coli
1. Kozak Sequence
2. 6xHis-Tag
3. 10aa GS Linker
4. TEV Cut site
5. GLP-1R 
   sequence 
   codon 
   optimized 
   for E. E. coli
   pET28:GFP
Zoe Petroff 5650

  1. [1] J. J. Holst, “The physiology of glucagon-like peptide 1,” Physiol Rev, vol. 87, no. 4, pp. 1409–1439, Oct. 2007, doi: 10.1152/physrev.00034.2006.