Sponsored by:
H. Chaudhry, I. Burova, W. Yousaf, C. Davis, B. Seeley, B. Livingstone, C. de Cock, D. Goss
University of Warwick
Introduction
IRES
We began this project with a desire to introduce the world of RNA to iGEM. RNA is
useful to the synthetic biology community as it is multifunctional, catalytic and
dynamic. In the future, we hope that RNA systems become more commonplace as a
result of our contributions. We have created a toolkit of different RNA parts
with key functions; forming the foundation of what we hope is an RNA revolution. As
an embodiment of an RNA system, we focused on inhibition of Dipeptidyl peptidase-IV,
a form of treatment for Type 2 Diabetes.
An IRES (Internal Ribosome Entry Site) allows translation initiation
within an RNA strand, which we need as our replicon contains
multiple protein coding regions separated by structural nucleotide
sequences. We characterised two IRES sequences, derived from
Encephalomyocarditis virus (EMCV) and from the NF-kappaB
repressor factor (NKRF) untranslated region.
Neomycin
Figure 1. IRES Testing Module.
The testing module was used for IRES
characterisation in Huh 7.5 and HeLa cells.
A split neoymcin gene confers kanamycin and
geneticin resistance to E. coli and eukaryotic
cells respectively. This allows selection for cotransformants without using multiple antibiotics.
Figure 2. Comparison of EMCV vs NKRF
IRES efficacy.
Comparison of the EMCV and NKRF IRES in
HeLa and Huh 7.5 cells.
Figure 3. Mechanism of Neomycin action.
Neo-α and neo- β interact through a leucine zipper to
reconstitute a heterodimer that confers antibiotic
resistance [1].
RNA
Promoter
MS2 Box
Neomycin
IRES
GFP Output
siRNA
IRES
DICER
MS2
Coat Protein
P2A
RdRp
RNA
Promoter
RNA Promoters and RdRp
RdRp catalyses the replication of RNA from an RNA template. The replication process begins with the
positive RNA strand. The RdRp binds to the conserved 3’ UTR (which we liken to an RNA Promoter) and
replicates a full length negative strand intermediate. The negative strand is then used to generate
positive RNA strands in the same manner, binding to the 5‘ RNA Promoter which is now the new 3’ UTR.
The process then repeats.
Figure 7. Aptazyme efficiency in HeLa and Huh 7.5 cells.
To test the efficiency of splicing and thus the potential of aptazymes as RNA
kill switches, mRNA containing an aptazyme and GFP was transfected into
Figure 6. Mechanism of aptazyme action.
cells and fluorescence was measured after 24 hours. We observed a
Theopylline introduction induces a conformational
theophylline induced 22% reduction of fluorescence in Huh7.5 cells. In
change which activates the aptazyme, leading to RNA HeLa cells however, more testing is required.
cleavage. [3]
Plasmids in E. coli Using a Single Selective Marker.
DICER
Figure 4. Folding Analysis of siRNA Output.
Secondary structure of the siRNA sequences in the positive and negative sense. In the negative
sense, the stem attracts DICER to produce siRNA segments. This is not the case in the positive sense.
1.
1. Schmidt, C., Shis, D., Nguyen-Huu, T. and Bennett, M. (2012). Stable Maintenance of Multiple
2.
3.
Figure 9. Illustration of RdRp replicating mechanism.
Our RdRp part is derived from the Hepatitis C Virus Con1 strain [4]. To test RNA promoter activity, a
plasmid encoding NS5B RdRp was introduced under the expression of a T7 promoter. A second plasmid
harbouring promoters with reverse GFP was also introduced in E. coli. Induction of RdRp production led
to a near ~2 fold increase in fluorescence in E. coli, relative to non-induced control bacteria, RdRp
produced the positive strand RNA from its reverse complement. By allowing the creation of RNA replicons,
RdRp could allow the creation of RNA only systems.
2. Inamoto, T., Yamada, T., Ohnuma, K., Kina, S., Takahashi, N., Yamochi, T., Inamoto, S., Katsuoka,
Y., Hosono, O., Tanaka, H., Dang, N. and Morimoto, C. (2007). Humanized Anti-CD26
Figure 5. Ordinary vs Stochastic Time Delay Differential Equation Solution.
The time delay differential equations for our 3’ UTR experiment, solved numerically
(in red) and stochastically (in blue, averaging 100 repeats) with the solver we created.
The MS2 Coat Protein and MS2 box are used
as translation repression elements. These
regulate RdRp production through a negative
feedback loop as RdRp and MS2 are
co-translated (via a P2A linker).
An aptazyme is self-cleaving RNA; activated
by a small molecule. We use aptazymes in
our construct as a theophylline activated
RNA kill switch.
References
Time
Aptazyme
siRNA, small interfering RNAs, are produced when DICER recognises a 20-25 bp stem of
dsRNA, then processes them into ~22 bases long ssRNAs. These are recruited by the RISC
complex, and used as a template to target mRNA, which is then cleaved. The siRNA targets
the 3’UTR of the DPP-IV protein [2]. We designed the sequence so that the negative strand
would form a stem loop but the positive one would not, by exploiting the fact that G-C
and G-U base pairings are possible (whereas on the complementary strand, C will not pair
with A).
MS2 Box and MS2 Coat Protein
Aptazyme
Modelling
In addition to modelling of the individual experiments conducted,
we looked to further advance the modelling efforts of future iGEM
teams. To this end we created a toolbox of MATLAB programs that
allow for more mathematically sophisticated sytems to be solved.
We created solvers that can handle time-delay implementations,
as well as stochastic systems, but also stochastic time-delay
systems. Ultimately, this means that systems that capture a greater
essence of realism can be incorporated into iGEM projects. We also
created a program to aid in characterisation of parts, in that it helps
analyse results obtained from a Tecan plate reader. All programs
mentioned are included on our wiki.
siRNA
Monoclonal Antibody as a Treatment for Malignant Mesothelioma Tumors.
3. Wieland, M. and Hartig, J. (2008). Improved Aptazyme Design and In Vivo Screening Enable
Riboswitching in Bacteria.
4. Lohmann V, e. (1997). Replication of subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell
Line.
Figure 8. RdRp activity.
Fluorescence of E coli cells with constitutive expression of the reverse complement of GFP-RNA promoter, with or without induction of RdRp, at various ODs. Error bars represent standard deviation of
three biological replicates.