Parts - Lethbridge HS iGEM

Schematic of Our Sa-mRNA Construct

The start codon (>) is always AUG but the stop codons (x) can be either UAA, UAG, UGA. These are important for the ribosome to start and stop translation so the proteins being made have a defined sequence.
Cut sites (v), otherwise called restriction enzyme recognition sites, are specific sequences that restriction enzymes bind to and cut, hence the colloquial name. Although there are a few more than depicted in the above schematic, the ones shown are most important. They were intentionally added to facilitate easier replacement of the aptamer (A) and protein of interest. Each site is unique, so the corresponding restriction enzyme will only cut the one spot in the sequence.
From left to right (3' to 5'): T7P = T7 polymerase promoter, 5' UTR = 5' conserved untranslated region, NSP1-4 = nonstructural proteins 1-4, SGP = subgenomic promoter, A = aptamer (interchangeable), KS = Kozak sequence, hI = human insulin, L= linker, GFP = green fluorescent protein, 3' UTR = 3' conserved untranslated region, PA = polyA tail.

Basic Parts

Name
Type
Description
Source(s)

BBa_J64997
Basic / Regulatory
T7P
iGEM Registry of Biological Parts

BBa_K4351001
Basic / Regulatory
5' CSE
GenBank: DQ322637.1

BBa_K4351003
Basic / Regulatory
3'CSE
GenBank: DQ322637.1

BBa_K4351004
Basic / Regulatory
VEEV subgenomic promoter
GenBank: DQ322637.1

BBa_K4351005
BBa_K4351006
BBa_K4351007
BBa_K4351008
Basic / Coding
VEEV NSP1-4
GenBank: DQ322637.1

BBa_K4351009
Basic / Regulatory
Kozak sequence
1: McClements M. et al., (2021)

BBa_K4351000
Basic / Regulatory
Glucose binding RNA aptamer 1
4: Yang KA, et al (2014)

BBa_K4351011
Basic / Regulatory
Glucose binding RNA aptamer 2
5. Ma Y, et al (2018)

BBa_K4351012
Basic / Regulatory
A scramble of the glucose binding aptamer
BBa_K4351000

BBa_K4351013
Basic / Regulatory
Theophylline RNA aptamer
6. Rankin CJ, et al (2006)

BBa_K4351014
Basic / Regulatory
Insulin binding RNA aptamer 1
2: Taghdisi SM, et al (2015)

BBa_K4351015
Basic / Regulatory
Insulin binding RNA aptamer 2
3: Wu Y, et al (2019)

BBa_K4351016
Composite / Regulatory
Insulin binding RNA aptamer 1 + RNA Mango
2: Taghdisi SM, et al (2015)

BBa_K4351017
Composite / Regulatory
Insulin binding RNA aptamer 2 + RNA Mango
3: Wu Y, et al (2019)

BBa_K4351018
Composite / Regulatory
Glucose binding RNA aptamer 1 + RNA Mango
4: Yang KA, et al (2014)

BBa_K4351019
Composite / Regulatory
Glucose binding RNA aptamer 2 + RNA Mango
5. Ma Y, et al (2018)

BBa_K4351020
Composite / Experimental control
A scramble of the glucose binding aptamer 1 + RNA Mango
N/A

BBa_K4351021
Composite / Regulatory
Theophylline RNA aptamer + RNA Mango
6. Rankin CJ, et al (2006)

BBa_K4351010
Removed Sn34 sequence from
BBa_K3001024
Basic / Reporter
RNA mango aptamer was combined with the other aptamers for testing their function
12. Dolgosheina, et al (2014)
iGEM Registry of Biological Parts

BBa_K4351022
Basic / Coding
A fusion protein coding sequence made of the combined human insulin and GFP sequences connected by a linker
GenBank: BT006808.1
7: Chen, et al (2013)
GenBank: U73901.1

BBa_K4351023
Composite / Device
The combination of all of the above sequences into one final sa-mRNA product for expressing an insulin-GFP fusion protein. Has a polyA tail sequence at the 5' end.

Acquiring Sequences
Sequences from the Venezuelan Equine Encephalitis Virus (VEEV) genome required approval from the iGEM safety committee. The sequences we found were cross-referenced to an “off-the-shelf” sa-mRNA sequence provided by Dr. Blakney. The sa-mRNA sequence could not be ordered as g-blocks from IDT, a generous sponsor of iGEM, without additional paperwork.

Testing
This sa-mRNA sequence can be inserted into a plasmid with a traditional antibiotic resistance cassette for selective cloning. This way the DNA sequence can be amplified and purified for use as an in vitro transcription (IVT) template.
The best modes of testing sa-mRNA success start with transfecting the appropriate mammalian cell lines with sa-mRNA purified from an IVT. For human proteins of interest a human embryonic kidney cell line like HEK293T would provide a sufficient environment for testing expression. Translation could be measured using flow cytometry, which would detect fluorescence of the insulin-GFP fusion protein.

Sequence Descriptions
We intended to treat type 1 diabetes in humans but soon learned that beloved family pets might also benefit from our project. Our project Dia-Beatable uses a self-amplifying messenger RNA consisting of multiple sequences. In the future, sequences will have to be altered to be species-specific, depending on the target patient.

A T7 polymerase promoter is a part of the sa-mRNA to allow for amplification of the RNA sequence using in vitro transcription. This means the sa-mRNA can be manufactured at high concentration in the lab.

The 5' and 3' Conserved Sequence Elements, subgenomic promoter, and non-structural proteins were all acquired from a VEEV genome. Specifically, the VEEV replicon vector YFV-C1 submitted by Shustov, A.V., and Frolov, I.V. to the NIH National Library of Medicine GenBank (GenBank: DQ322637.1). NSP1-4 are translated as one large protein which breaks into several individual proteins through autolytic activity [8]. Together, they function as two different RNA-dependent RNA polymerases, first making complementary negative sense RNA, then more positive sense RNA that the ribosome can use as templates for translation [9]. The subgenomic promoter will encourage the RNA-dependent RNA polymerase to produce an additional mRNA containing only the GOI [9].

With the replicating nature of our proposed treatment, there must be a control mechanism present; the insulin-dependent RNA aptamer takes on this role. The RNA aptamer of Dia-Beatable is receptive to insulin levels. When high levels of insulin are detected in the cytoplasm, insulin will bind the RNA aptamer, preventing further translation of insulin by changing the 3D structure of the ribosome binding site (see modeling page for more details). Translation of insulin from the sa-mRNA and subgenomic RNA will resume once insulin levels are low.
Of course, before we decided on an insulin RNA aptamer, we had been working with two glucose binding RNA aptamers (experimental), a scramble of a glucose aptamer sequence (negative control), and a theophylline binding RNA aptamer (positive control). The parts were tested for function using fluorescence spectrophotometry. We measured the change in fluorescence over time. Theoretically, when the ligand (glucose, insulin, or theophylline) binds to the aptamer the downstream RNA structure changes to allow for incorporation of a fluorophore called thiazole orange (TOI) that further increases fluorescence.
Our insulin binding aptamer has been ordered but has not yet been tested. It requires a test that measures for translation inhibition, which could be done by measuring the translation of a downstream fluorescent protein. Theoretically, fluorescence would cease to increase in the presence of insulin. Further optimization is needed to tailor the dissociation constant of insulin binding to the aptamer and further modeling is needed to determine a safe level of insulin inside of an individual cell required to sustain a basal insulin level of 15 ∓4.8 µU/ml in the body [10].

For the Kozak Sequence, we chose 5' - GCCGCCACC - 3'. McClements M. et al., (2021) used this sequence in a plasmid to transfect a human cell line and showed it to function as predicted. The Kozak Sequence, although not necessary, is used in our project due to its immense influence in increasing translation efficiency [1].

We have decided to use the full human insulin gene sequence for the preliminary stages of our project, acquired from the NIH GenBank (GenBank: BT006808.1). With this insulin being produced in the body, there is no need for sequence modification that aids in stability and purification like those required by commercial insulin. In native insulin, there are three chains; A, B, and C. The connecting (C) chain orients the A and B chains in such a way that cysteine bridges can form before the C chain is cleaved off and discarded [11].
It is possible to test for presence of insulin using Western Blotting. However, by creating a fusion protein with insulin and green fluorescent protein (GFP), translation of our POI could also be rapidly tested using flow cytometry. This technique would measure fluorescence output from GFP within cells. We added a linker sequence - ggcagcgcgggcagcgcggcgggcagcggcgaattt- from Chen, et al (2013) specialized for creating GFP fusion proteins [7]. The GFP sequence was retrieved from NIH GenBank (GenBank: U73901.1).

References
[1] McClements, M. E., Butt, A., Piotter, E., Peddle, C. F., & MacLaren, R. E. (2021). An analysis of the Kozak consensus in retinal genes and its relevance to gene therapy. Molecular vision, 27, 233–242.

[2] Seyed Mohammad Taghdisi, Noor Mohammad Danesh, Parirokh Lavaee, Ahmad Sarreshtehdar Emrani, Mohammad Ramezani & Khalil Abnous (2015) Aptamer Biosensor for Selective and Rapid Determination of Insulin, Analytical Letters, 48:4, 672-681, DOI: 10.1080/00032719.2014.956216

[3] Yao Wu, Beksultan Midinov, and Ryan J. White. ACS Sensors 2019 4 (2), 498-503

[4] Yang KA, Barbu M, Halim M, Pallavi P, Kim B, Kolpashchikov DM, Pecic S, Taylor S, Worgall TS, Stojanovic MN. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors. Nat Chem. 2014 Nov;6(11):1003-8. doi: 10.1038/nchem.2058. Epub 2014 Sep 28. PMID: 25343606; PMCID: PMC4339820.

[5] Y. Ma, Y. Mao, Y. An, T. Tian, H. Zhang, J. Yan, Z. Zhu and C. J. Yang, Analyst, 2018, DOI: 10.1039/C8AN00010G.

[6] Rankin, C. J.; Fuller, E. N.; Hamor, K. H.; Gabarra, S. A.; Shields, T. P. (2006). A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer. Nucleosides, Nucleotides and Nucleic Acids, 25(12), 1407–1424. doi:10.1080/15257770600919084

[7] Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69. doi: 10.1016/j.addr.2012.09.039. Epub 2012 Sep 29. PMID: 23026637; PMCID: PMC3726540.

[8] Kääriäinen L, Ahola T. Functions of alphavirus nonstructural proteins in RNA replication. Prog Nucleic Acid Res Mol Biol. 2002;71:187-222. doi: 10.1016/s0079-6603(02)71044-1. PMID: 12102555; PMCID: PMC7133189.

[9] Blakney AK, McKay PF and Shattock RJ (2018) Structural Components for Amplification of Positive and Negative Strand VEEV Splitzicons. Front. Mol. Biosci. 5:71. doi: 10.3389/fmolb.2018.00071

[10] Bagdade JD, Bierman EL, Porte D Jr. The significance of basal insulin levels in the evaluation of the insulin response to glucose in diabetic and nondiabetic subjects. J Clin Invest. 1967 Oct;46(10):1549-57. doi: 10.1172/JCI105646. PMID: 6061732; PMCID: PMC292903.

[11] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013 Jan 1;9(1):25-53. PMID: 22974359; PMCID: PMC3934755.

[12] Dolgosheina EV, Jeng SCY, Panchapakesan SSS, Cojocaru R, Chen PSK, Wilson PD, Hawkins N, Wiggins PA, and Unrau PJ. ACS Chemical Biology 2014 9 (10), 2412-2420 DOI: 10.1021/cb500499x

Name	Type	Description	Source(s)
BBa_J64997	Basic / Regulatory	T7P	iGEM Registry of Biological Parts
BBa_K4351001	Basic / Regulatory	5' CSE	GenBank: DQ322637.1
BBa_K4351003	Basic / Regulatory	3'CSE	GenBank: DQ322637.1
BBa_K4351004	Basic / Regulatory	VEEV subgenomic promoter	GenBank: DQ322637.1
BBa_K4351005 BBa_K4351006 BBa_K4351007 BBa_K4351008	Basic / Coding	VEEV NSP1-4	GenBank: DQ322637.1
BBa_K4351009	Basic / Regulatory	Kozak sequence	1: McClements M. et al., (2021)
BBa_K4351000	Basic / Regulatory	Glucose binding RNA aptamer 1	4: Yang KA, et al (2014)
BBa_K4351011	Basic / Regulatory	Glucose binding RNA aptamer 2	5. Ma Y, et al (2018)
BBa_K4351012	Basic / Regulatory	A scramble of the glucose binding aptamer	BBa_K4351000
BBa_K4351013	Basic / Regulatory	Theophylline RNA aptamer	6. Rankin CJ, et al (2006)
BBa_K4351014	Basic / Regulatory	Insulin binding RNA aptamer 1	2: Taghdisi SM, et al (2015)
BBa_K4351015	Basic / Regulatory	Insulin binding RNA aptamer 2	3: Wu Y, et al (2019)
BBa_K4351016	Composite / Regulatory	Insulin binding RNA aptamer 1 + RNA Mango	2: Taghdisi SM, et al (2015)
BBa_K4351017	Composite / Regulatory	Insulin binding RNA aptamer 2 + RNA Mango	3: Wu Y, et al (2019)
BBa_K4351018	Composite / Regulatory	Glucose binding RNA aptamer 1 + RNA Mango	4: Yang KA, et al (2014)
BBa_K4351019	Composite / Regulatory	Glucose binding RNA aptamer 2 + RNA Mango	5. Ma Y, et al (2018)
BBa_K4351020	Composite / Experimental control	A scramble of the glucose binding aptamer 1 + RNA Mango	N/A
BBa_K4351021	Composite / Regulatory	Theophylline RNA aptamer + RNA Mango	6. Rankin CJ, et al (2006)
BBa_K4351010 Removed Sn34 sequence from BBa_K3001024	Basic / Reporter	RNA mango aptamer was combined with the other aptamers for testing their function	12. Dolgosheina, et al (2014) iGEM Registry of Biological Parts
BBa_K4351022	Basic / Coding	A fusion protein coding sequence made of the combined human insulin and GFP sequences connected by a linker	GenBank: BT006808.1 7: Chen, et al (2013) GenBank: U73901.1
BBa_K4351023	Composite / Device	The combination of all of the above sequences into one final sa-mRNA product for expressing an insulin-GFP fusion protein. Has a polyA tail sequence at the 5' end.