Team:BGU Israel/Parts
Parts Overview
OriginALS is proud to present its registry parts! These biobricks are integral to our two-dimensional ALS therapy and maybe one day to another synthetic biology project!
For more information click on the part link:
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Short description |
NickName |
Part link |
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Tissue inhibitor of metalloproteinases-1 promoter (pTIMP1) |
pTIMP1 |
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gRNA#1 for SaCas9 targeting of IKKβ under U6 promoter |
U6 g1 |
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gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g2 |
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gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g5 |
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gRNA#6 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g6 |
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Microglia specific promoter pF4/80 |
pF4/80 |
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Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) with a point mutation |
pSTEAP4 +mut |
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Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) |
pSTEAP4 |
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Synthetic minimal adenovirus major late promoter (pMLPm) |
pMLP |
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Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm |
RGR gRNA |
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pSTEAP4 RGR gRNA (composite part) |
pSTEAP4 RGR gRNA |
Best basic part - BBa_K2538005
pTimp1 (tissue inhibitor of metalloproteinases-1 promoter) is highly expressed in A1 reactive astrocyte cells. We were able to confirm the promoter strength in C8D30 (A1 astrocyte cell line) through a dual luciferase promoter assay (Fig 1):
Figure 1: The activity of pTimp promoters in reactive astrocyte cell line (C8-D30). Luciferase reporter assay demonstrating transcriptional activation mediated by promoter of pTimp1 in C8-D30 cells. The pTimp promoter shoed 27.5-fold increased activity, as compared to the control (empty vector). Relative luciferase expression results are presented after normalization to Renilla activity and represents the mean ± standard deviation of three independent experiments.
Then, we used this promoter to specifically express CRISPR dCas9 VP64 in order to activate the synthetic minimal adenovirus major late promoter (pMLPm) that will express reverse Caspase3, by doing so we will be able to induce apoptotic death only in reactive astrocytes and not in the resting cells. By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival.
mCherry reporter protein is also expressed under the control of the pMLPm promoter, it is fused to the exogenous caspase3 with Thoseaasigna virus 2A (T2A) peptide that is cleaved during translation. mCherry expression was detected in fluorescent microscope (Fig 2):
Figure 2: Images of mCherry in C8-D30 cells 24 hours post co-transfection with lenti:pTimp1-VP64 and pSynt. A – Fluorescent microscope. B – Bright field.
Best composite part - BBa_K2538011
In ALS disease, under gliosis conditions astrocyte cells are transformed from their resting to their reactive form, this transformation includes the induction of several genes including Six Transmembrane Epithelial Antigen of Prostate 4 gene by pSteap4 promoter.
In this composite part the pSteap4 promoter regulates the expression of gRNA which its spacer is complementary to three different loci in the synthetic minimal adenovirus major late promoter (pMLPm). The promoter will be active only under the condition that gRNA-dCas9-VP64 complex promotes downstream transcription. When the transcription factor VP64 (an engineered tetramer of the herpes simplex VP16 transcriptional activator domain) is fused to dCas9 enzyme it can be guided to a specific location in the genome, in this manner we can exploit it to promote downstream translation.
The gRNA-dCas9-VP64 complex will target the synthetic minimal adenovirus major late promoter (pMLPm) and promotes the expression of exogenous reverse caspase3, that in contrast to the endogenous caspas3 will be activated after transcription due to its autocatalytic processing, meaning that this enzyme will trigger an apoptotic signal that will lead to apoptotic death (caspase3 is responsible for chromatin condensation and DNA fragmentation).
mCherry reporter protein is also expressed by the pMLPm promoter, it is fused to the exogenous reverse caspase3 with Thoseaasigna virus 2A (T2A) peptide that is cleaved during translation.
mCherry expression was detected in fluorescent microscope (Fig 2).
We used the pSteap4 promoter to specifically express gRNA that will connect to dCas9 enzyme and VP64 transcription factors, together our system will target and activate the synthetic promoter pMLPm that will express reverse Caspase3, by doing so we will be able to induce apoptotic cell death only in reactive astrocyte and will discriminate against resting cells. By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival.
Best collection
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no available treatment. The BGU-IGEM team aims to develop a system that will ultimately prolong survival of ALS patients by targeting microglia and reactive astrocytes, which are both non-neuronal cells that directly contribute to motor neuronal damage.
The "OriginALS" collection was designed to fulfill our therapeutic approach:
- Inhibition of toxic pro-inflammatory cytokines secretion in microglia cells.
- Promoting intrinsic apoptotic signal in reactive astrocytes and preventing their toxic effect on motor neurons.
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Short description |
NickName |
Part link |
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Tissue inhibitor of metalloproteinases-1 promoter (pTIMP1) |
pTIMP1 |
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gRNA#1 for SaCas9 targeting of IKKβ under U6 promoter |
U6 g1 |
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gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g2 |
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gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g5 |
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gRNA#6 for SaCas9 targeting of IKKβ under U6 promoter |
U6-g6 |
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Microglia specific promoter pF4/80 |
pF4/80 |
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Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) with a point mutation |
pSTEAP4 +mut |
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Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) |
pSTEAP4 |
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Synthetic minimal adenovirus major late promoter (pMLPm) |
pMLP |
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Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm |
RGR gRNA |
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pSTEAP4 RGR gRNA (composite part) |
pSTEAP4 RGR gRNA |
Using modified genome editing technique, we built a system that specifically targets toxic astrocytes and prevents the formation of new ones which hopefully will slow down the progression of the disease. As the reactivity of microglia and astrocytes is common to many neurodegenerative diseases, our novel approach could be expanded to other neurodegenerative diseases. In this unique collection you can find our specific promoters and gRNA.