Part:BBa_K5237024
TRE-minimal promoter- firefly luciferase
This part contains the tetO binding site (BBa_K5237019) , a minimal promoter and a firefly luciferase gene. With a VP64 coming in close proximity to the minimal promoter transcription factors are recruited, initiating expression of firefly luciferase. The described mechanism is utilized in our enhancer hijacking assay for prove of Cas stapling.
Contents
Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene
regulation,
cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture
remain limited, rendering it challenging to explore the full potential of the
3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
toolbox based on various DNA-binding proteins to address this issue.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding
proteins
include our
finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
and can be further engineered to create alternative, simpler and more compact staples.
(ii) As functional elements, we list additional parts that enhance the functionality of our Cas and
Basic staples. These
consist of
protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo.
Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
with our
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of our custom
readout
systems. These include components of our established FRET-based proximity assay system, enabling users to
confirm
accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
in mammalian cells.
The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed
exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in
the
collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
own custom Cas staples, enabling further optimization and innovation.
Our part collection includes:
| DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly. | ||
| BBa_K5237000 | fgRNA Entry vector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple |
| BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple |
| BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close proximity |
| BBa_K5237004 | Staple subunit: Oct1-DBD | Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237005 | Staple subunit: TetR | Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237006 | Simple staple: TetR-Oct1 | Functional staple that can be used to bring two DNA strands in close proximity |
| BBa_K5237007 | Staple subunit: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237008 | Staple subunit: rGCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237009 | Mini staple: bGCN4 | Assembled staple with minimal size that can be further engineered | Functional elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications |
| BBa_K5237010 | Cathepsin B-cleavable Linker: GFLG | Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples |
| BBa_K5237011 | Cathepsin B Expression Cassette | Expression Cassette for the overexpression of cathepsin B |
| BBa_K5237012 | Caged NpuN Intein | A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs |
| BBa_K4643003 | incP origin of transfer | Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery | Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity |
| BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I | Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity |
| BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay |
| BBa_K5237019 | TetR Binding Cassette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 | Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker |
| BBa_K5237021 | NLS-Gal4-VP64 | Trans-activating enhancer, that can be used to simulate enhancer hijacking | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promotor, mCherry | Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker |
| BBa_K5237023 | Oct1 - 5x UAS binding casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay |
| BBa_K5237024 | TRE-minimal promoter- firefly luciferase | Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence overview
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 99
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 99
Illegal BamHI site found at 78
Illegal XhoI site found at 48 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 952
The tetracycline response element, part of the TetR family of regulators (TFRs), is central to the regulation of
antibiotic resistance genes, especially through its role in the Tet operon (TetO). In this system, the TetR protein
acts
as a repressor by binding to the TetO operator, inhibiting the expression of tetracycline resistance genes. Upon
binding
tetracycline or its analogs, TetR undergoes a conformational change, releasing TetO and allowing the transcription
of
target genes. This system is widely utilized in molecular biology as a controlled gene expression tool, particularly
in
inducible gene expression systems in both prokaryotic and eukaryotic cells (Cuthbertson and Nodwell (2013)).
Firefly luciferases are enzymes responsible for the bioluminescence seen in fireflies, catalyzing the oxidation of
luciferin in the presence of ATP, magnesium ions, and oxygen. This reaction produces light and is highly efficient,
with
little heat released, making it a popular tool in molecular biology for reporter assays. The gene encoding firefly
luciferase is widely used in research to monitor gene expression, quantify cellular ATP levels, and study
transcriptional activity due to its sensitivity and ease of detection (Xie et al. (2010))
We utilize the recognition site for plasmid to plasmid stapling with our Cas staples. A fgRNA targeting Tre and Oct1
on
the other plasmid, is the key factor in the Cas staple, bringing them together. When the transactivator, Gal4-VP64,
binds as well we have transactivation as a readout for functioning staples.
The cloning strategy designed for TetO allows for the easy assembly of repetitive repeats. It follows the procedure
outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI and
XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be linearized
with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated into
the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility. This
process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the oligos.
For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some
limitations regarding sequence depository, the binding cassette is flanked by SalI and XhoI, and the top and bottom
oligos with the fitting overhangs are annotated.
We were able to show our enhancer plasmid to work great with the Cas staples and the reporter plasmid. For the whole
assay, an enhancer plasmid and the reporter plasmid was used. The reporter plasmid has firefly luciferase behind
several repeats of a Cas9 targeted sequence. The enhancer plasmid has the Oct1 being targeted by Cas12a. By
introducing a fgRNA staple (BBa_K5237000) and a Gal4-VP64 (BBa_K5237021), expression of the luciferase is induced
(Fig. 2 A).
Cells were again normalized against ubiquitous renilla expression.
Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 2 B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher
expression of the reporter gene. These results suggest an extension of the linker might lead to better
transactivation when hijacking an enhancer/activator.
Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor
for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. https://doi.org/10.1038/srep109072. Usage and Biology
3. Assembly and part evolution
4. Results
5. References