iGEM IOANNINA designed IRIS, a bacterial machine that is able to perform Photodynamic Therapy [PDT] in a simple,
yet futuristic way. Through IRIS Protoporphyrin IX [PpIX], a Photosensitizer widely utilized in classic PDT techniques,
is overexpressed in cancer cells, thus improving the efficiency of the treatment. As PpIX accumulates in the tumor site, light of a
specific wavelength is generated by the bacteria themselves.
The light occurs as an exogenous substrate, called methoxy e-coelenterazine, reacts with Rluc8 luciferase, a
mutated variant of Renilla reniformis’ enzyme Rluc, expressed by IRIS.
reacts with Rluc8 luciferase, an enzyme expressed by IRIS.
Although it seems unreal, IRIS’s mechanism of action can be separated in three basic parts:
Protoporphyrin IX production
Aminolevulinic acid is not a photosensitizer, but a metabolic precursor of protoporphyrin IX (PpIX),
which is a common photosensitizer.
The synthesis of 5-ALA is normally tightly controlled by feedback inhibition of the enzyme,
ALA synthetase, presumably by intracellular heme levels.
ALA, when provided to the cell, bypasses this control point and results in the accumulation of PpIX.
[1]
According to the presumed mechanism of action, photosensitization occurs through the metabolic conversion of
5-ALA to protoporphyrin IX (PpIX).
When exposed to light of appropriate wavelength and energy, the accumulated
PpIX produces a photodynamic reaction, a cytotoxic process
dependent upon the simultaneous presence of light and oxygen.
The absorption of light results in an excited state of the porphyrin molecule,
and subsequent spin transfer from PpIX to molecular oxygen generates
singlet oxygen, which can further react to form superoxide and hydroxyl
radicals. [2]
The biosynthesis of ALA is tightly regulated by the end product,
heme, but the regulation mechanism of the heme biosynthesis pathway is
complex and understudied [3] .
Zhang, J., Kang, Z., Chen, J. et al. studied and optimized the
heme biosynthesis pathway for the production of 5-aminolevulinic acid in
Escherichia coli, providing significant results and insight into this topic. After checking the effects of overexpression of both the
downstream genes (hemB, hemC, hemD, hemE, hemF, hemG and hemH) and the upstream genes (hemA and hemL),
the team concluded that the highest 5-ALA accumulation occurs through combinatorial overexpression of hemA, hemL, hemD and hemF.
[4]
Furthermore, the results suggested that high-level overexpression of the genes hemAs, hemL, hemF, and
moderate overexpression of the
downstream gene hemD are favorable to ALA accumulation.
Specifically, due to HemA and HemL forming a tight complex, with a 1:1 ratio,
to quickly catalyze glutamyl-tRNA to ALA, the upstream genes hemL and hemAs
were co-overexpressed with the downstream gene hemF. [4]
Based on these results, we decided to co-overexpress hemAs, hemL, hemF in the same high-copy-plasmid to achieve high-level overexpression.
The genes hemAs and hemL are expressed under the same T7 promoter and hemF is expressed under a second T7 promoter in the same plasmid.
Moderate over-expression of hemD is achieved by integrating
it into a low-copy number plasmid. See figure down below:
In addition, a threonine/homoserine exporter was identified to be encoded by rhtA gene in E. coli,
which exports 5-ALA due to its broad substrate specificity.
[5] ALA has similar chemical structure to glycylglycine and its physical properties are close to native amino acids
carrying uncharged side chains, thus making
it possible to be exported via this threonine/homoserine exporter expressed by rhtA gene.
Oxygen release
One of the biggest obstacles in IRIS’s way is the
lack of oxygen in the hypoxic tumor regions.
Hypoxia is a problem, as molecular oxygen
is required for the production of ROS in Photodynamic Therapy.
Catalase is an enzyme found in nearly all living organisms, which catalyzes the decomposition of hydrogen peroxide to water and oxygen.
It has been indicated that overexpression of catalase in E. coli gives them increased PDT capacity. [6]
In this process, catalase turns H2O2 into O2 in tumor site to enhance the effect of PDT.
![](https://static.igem.wiki/teams/4807/wiki/design-images/oxygen-rel.png)
Light generation
The most common light sources in traditional PDT techniques are asers, light-emitting diodes (LEDs) and lampsl.
[7]
IRIS’s groundbreaking indication is that the light needed for PDT can be endogenously
produced by the bacteria themselves, making external light administration unnecessary.
PpIX has five absorption wavelength peaks: 410 nm, 510 nm, 545 nm, 580 nm, and 630 nm. [8]
Blue light, around 400 nm, is usually employed as a light source for PDT [8],
so we wanted to employ a luciferin-luciferase pair that produces light at around 410nm.
One R. reniformis luciferase mutation variant (RLuc8) has greater stability and emits more light than the native enzyme (RLuc).
[9] Prolume Purple or methoxy e-Coelenterazine (Me-O-e-CTZ) is a synthetic analogue of e-Coelenterazine with an additional methoxy group,
shifting the emission maximum from 475 nm to around 410 nm. [10]
IRIS expresses Rluc8, which subsequently reacts with methoxy e-Coelenterazine, emitting light around 410nm, thus eliminating the need for delivering light exogenously.
![](https://static.igem.wiki/teams/4807/wiki/design-images/coelenterazine-2-ff.gif)
HiFi DNA Assembly
HiFi DNA Assembly
Our team considered different assembly approaches to build our constructs. We finally decided to use HiFi DNA Assembly,
a simple and fast cloning procedure that utilizes a high-fidelity polymerase for the assembly of multiple DNA fragments,
regardless of fragment length or end compatibility.
Its main advantage is that it is based on a one-step-cloning model with a relatively simple master-mix format, it doesn’t
require any complicated insert or primer designing and it removes 3´- and 5´-end mismatches.
The one-step-cloning includes three different enzymes that work together in the same buffer:
1. The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region)
2. The polymerase fills in gaps within each annealed fragment.
3. The DNA ligase seals nicks in the assembled DNA, resulting in a double-stranded fully sealed DNA molecule.
Overview of HiFi DNA Assembly
Our vectors were linearized through digestion by restriction enzymes and our inserts were PCR-generated.
Restriction enzyme-treated vectors can have 5´ overhangs, 3´ overhangs or blunt ends.
One of the unique features of this Assembly Master Mix is its ability to remove both 3´ and 5´ end flap sequences upon fragment assembly.
T5 Exonuclease removes the bases from the 5-prime end, generating a 3-prime overhang. After that, single-stranded 3-prime ends can anneal,
the DNA polymerase fills in the occurring gap and DNA ligase joins the adjacent fragments.
In that way, the 3´- and 5´-end flap sequences occurring after a particular restriction enzyme digestion are removed.
This allows fragments generated by restriction enzyme digestion to assemble while eliminating the remaining restriction site sequences on both
the 5´ and 3´ ends in the fragment junction.
Primer Design
The first part of HiFi DNA Assembly is the Primer design. The basic rules we followed to design our primers are as follows:
The entire overlap sequence must originate from the vector sequence and must be added to primers that will be used to amplify the insert.
The overlap region of the forward primer for the gene of interest (orange) should line up with the 3´ end of the overhang on the vector’s left arm.
This primer should also include a gene-specific sequence at its 3´-end (gray).
Primer design - HiFi DNA Assembly
Clonetegration
Clonetegration
IRIS’s chromosomal DNA is also synthetically engineered. Clonetegration is a commonly used method for editing bacterial chromosomal DNA and incorporating gene
inserts into it.
Clonetegration is a two-plasmid system designed to integrate a gene into a specific site within the bacterial chromosome.
This approach combines DNA Cloning/assembly and chromosomal integration into a single procedure.
Clonetegration relies on bacteriophage-derived integrases, which are naturally involved in recombination processes between specific DNA sequences.
These integrases typically mediate recombination between attB sequences in the bacterial chromosome and an
attP sequence found on bacteriophage.
These attP sequences are incorporated into a plasmid, so they direct the integration of that plasmid into the chromosomal
attB sequence.
Clonetegration comprises two key plasmids:
OSIP plasmid
E-FLP plasmid
OSIP Plasmid:
OSIP is practically the plasmid responsible for the integration of the gene of interest in the bacterial chromosomal DNA. It is composed of two modules:
Integration/Propagation Module
Integration/Propagation Module
This module includes the following components:
Site-specific Recombinase Gene:
This gene is controlled by the lamda Cl ts repressor facilitating
precise site-specific recombination between attP and attB sequences.
Attp Attachment Sequence:
This sequence guides the integration of the plasmid into the bacterial chromosome
Antibiotic Selection Marker:
The marker is essential for the selection of the transformed cells
R6Kγ Origin of Replication:
This origin functions exclusively in strains that carry the pir gene. It permits the propagation of the plasmid
in pir+ stains and allows the stable integration of the plasmid into the
chromosome of the strain that lacks the pir gene.
FRT Sites:
These strains flank the integration/propagation module serving as recognition sites for the pirFillipase (FLP)
recombinase enzyme which facilitates the excision of this module from the bacterial chromosome.
Cloning Module
This module includes the following components:
Site-specific Recombinase Gene:
This gene is controlled by the lamda Cl ts repressor facilitating
precise site-specific recombination between attP and attB sequences.
Split Multiple Cloning Site (MCS)
this site accommodate BioBricks element BBa_I52002.
Mini pUC Origin:
it enables the production of high DNA yields of the unmodified plasmid
ccdB gene:
It functions as a negative selection marker because it expresses a toxin that binds to
the DNA gyrase enzyme.
DNA gyrase manages the DNA supercoiling by temporarily introducing double-stranded breaks in the DNA backbone,
and the interference by the ccdB toxin leads to cell death.
Genes integrated:
The genes Rluc8 and T7 RNA pol are inserted to the
cloning module in order to be integrated to the bacterial chromosome.
E-FLP plasmid:
The role of this vector is to express the Flp recombinase which recognize the Flippase Recognition Sites (FRT) sites and
catalyze the recombination between these sites. This recombination results in the excision of the
integration module from the bacterial chromosome.
Cloning Module
To enable IRIS to perform its diverse functions, it underwent a transformation from a typical E. coli bacterium into a significantly
altered engineered machine. To achieve this, our team incorporated three different plasmids housing the required genes, as well as
integrated two genes into IRIS's chromosomal DNA.
All the engineering was based on four distinct constructs:
Construct no. 1
Construct 1 - pTU2-A-RFP (p15A origin)/ hemD
Vector: pTU2-A-RFP
Gene inserted: hemD
To achieve a moderate expression of hemD gene, we employed pTU2-A-RFP, a low-copy-number plasmid, as the vector for this particular gene.
This vector features a p15A origin of replication and it is chloramphenicol resistant.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/ptu-a-rfp.png)
pTU2-A-RFP (p15A origin) vector
It lacks any T7 promoter/terminator or RBS sequences. To address this, we incorporated a T7 promoter, a T7 terminator, and an RBS, all originated
from pETDuet-1, in the final insert to serve as regulatory elements for controlling the expression of the hemD gene.
The vector was linearized through digestion by SacI restriction enzyme. We didn’t take into account any overhangs left by the enzyme when designing
primers and the actual insert, since HiFi DNA assembly removes both 3´ and 5´ end flap sequences in any case.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/hemd.png)
Construct 1 overview
Construct no. 2
Construct 2 - pET-29c(+) / hemL, hemAs, hemF
Vector: pET-29c(+)
Gene inserted: hemL, hemAs, hemF
In order to achieve high-level overexpression of hemL, hemAs and hemL we employed pET-29c(+), a high-copy-number plasmid, as the vector for these three genes.
This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is kanamycin resistant.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/pet-29c.png)
pET-29c(+) vector
In order to achieve the best results in 5-ALA accumulation, hemL and hemAs are expressed under the same T7 promoter,
while hemF is expressed under a separate promoter. [x] This means that two different transcription units,
both containing the necessary regulatory elements, must be integrated in one construct.
This vector already includes one T7 promoter, one RBS and one t7 terminator, so in order to regulate the second unit of our insert,
we integrated a T7 promoter, a T7 terminator and an RBS sequence, all originated from pETDuet-1.
The vector was linearized through digestion by two different restriction enzymes, BglII and SacI. These enzymes were specifically utilized to cut off a thrombin site. Once again,
we didn’t have to think about complementary one-stranded edges left by the enzymes, as blunt edges are always left during the HiFi DNA Assembly procedure.
To ensure their proper expression, each gene had to include a tag for its detection using Western blotting. Consequently, we incorporated an HA-tag and a
Myc-tag in our hemA and hemF inserts respectively. The hemL gene utilizes the S-tag provided by the vector.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/hemlasf.png)
Figure 10. Construct 2 overview
Construct no. 3
Figure 11. Construct 3 - pETDuet-1 / Catalase, rhtA
Vector: pETDuet-1
Gene inserted: Catalase, rhtA exporter
In order to achieve the overexpression of catalase and rhtA exporter, we employed pETDuet-1 as our carry out vector.
This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is ampicillin resistant.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/petduet-1.png)
Figure 12. pETDuet-1 vector
pETDuet-1 is a high copy number plasmid that features two T7 promoters and two RBS sequences. However, it contains only one T7 terminator.
As a result, each gene is expressed under its dedicated T7 promoter, but one of the inserted genes requires the inclusion of a T7 terminator
for proper regulation. The rhtA insert is the one carrying the extra T7 terminator.
In order to assemble our constructs, two cloning steps needed to be performed. Firstly, the vector was linearized through digestion with the BglII
restriction enzyme to allow the insertion of the catalase gene. Subsequently, another digestion with SacI was necessary to clone the rhtA exporter
gene into our vector.
In order to confirm our gene expression, an S-tag was incorporated in the catalase insert making it possible to detect through Western-Blotting.
The rhtA exporter utilizes a 6xHis-tag which is already an integral part of the plasmid.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/rhta-catalase.png)
Figure 13. Construct 3 overview
Construct no. 4
Figure 18. Construct 4
Vector: pOSIP-TT (P21)
Gene inserted: Rluc8
pOSIP is the vector mediating the insertion of Rluc8 in the bacterial chromosomal DNA as outlined in the clonetegration protocol.
Among the various plasmids within the OSIP family capable of performing this task, we chose pOSIP-TT (P21) due to its tetracycline resistance,
which was useful for simultaneous transformation with other plasmids.
This vector features a ColE1/pMB1/pBR322/pUC ori as well as a gamma replication origin from E. coli plasmid R6K and it is tetracycline resistant.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/posip-tt.png)
Figure 19. pOSIP-TT (P21) vector
pOSIP-TT (P21) lacks a T7 promoter, an RBS and a T7 terminator. To ensure the expression of our Rluc8 gene after its integration into the chromosomal DNA,
it's essential to incorporate these regulatory elements into our insert.
The vector was linearized through digestion with two different restriction enzymes, XhoI and EcorI. These enzymes were responsible for removing the ccdB toxin
and ColE1/pMB1/pBR322/pUC origin of replication in order to make the detection of the cloned vectors easier. Within this region, the Rluc8 gene was inserted,
complete with its regulatory elements.
It's unnecessary to incorporate a tag into our insert to verify the proper expression of the Rluc8 gene because Rluc8 is an enzyme that interacts with its substrate,
yielding a visible outcome.
![](https://static.igem.wiki/teams/4807/wiki/design-images/constructs-imgs/posip.png)
Figure 20. Construct 4 overview
Labeling our genes with different tags to enable the accurate detection of their expression via Western blot is a crucial step for optimizing our results.
We wanted each one of our genes to be labeled with a tag except the Rluc8 gene, as Rluc8 gives off its specific signal, fluorescence, when it interacts with its substrate, methoxy e-coelenterazine.
Taking into account our resources, depending on the available antibodies, we designed our initial tagging strategy employing three different
tags: 6-His, S-tag and HA-tag. Integrating the same tag in different proteins is not a problem, as long as their molecular
weights and lengths are different, making it possible to distinguish them via Western Blotting.
But according to our initial design, rhtA, hemD and hemF utilized the same tag, although their lengths and molecular weights are
similar. That would make them indistinguishable with Western-Blotting. Therefore, we decided to change hemF’s tag with a
Myc-tag and hemD’s tag with an S-tag.
The following table summarizes the Initial and Final Design for our tags:
Gene |
Length (bp) |
Molecular Weight (kDa) |
Initial Tag |
Final Tag |
rhtA |
888 |
33 |
6-His tag |
6-His tag |
Catalase |
1584 |
61 |
S-tag |
S-tag |
hemL |
1281 |
478 |
S-tag |
S-tag |
hemAs |
1254 |
37 |
HA-tag |
HA-tag |
hemF |
879 |
35 |
6-His tag |
Myc-tag |
hemD |
741 |
27 |
6-His tag |
S-tag |
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