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Team:Freiburg/parts/favorite parts
From 2013.igem.org
Our favorite BioBricks
No. 1: BBa_K1150020 - uniCAS Activator
Our uniCAS Activator
No. 2: BBa_K1150024 - uniCAS Histone Modifier
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| Figure X: CMV:dCas9-G9a (BBa_K1150024) dCas9 was fused via a 3 amino acid linker to G9a. The resulting fusion protein was flanked by NLS sequences and tagged by a HA epitope. The CMV promoter and BGH terminator were chosen to control gene expression. |
This device combines the dCas9 protein with the SET-domain of the murine histone methyltransferase G9a. dCas9 enables not only sequence specific, but also multiple targeting of any requested DNA sequence. Hence, coupling of dCas9 to the effector G9a allows for specific methylation of histone (Figure XXXX).
Such methylations are a hallmark of gene repression. One interesting fact about histone modification is the capability to spread the activity state over the surrounding chromatin via reader proteins. So the information of e.g. "repressed state" can, once specifically introduced, be propagated over a whole locus.
We included the G9a histone methylase to the uniCAS toolkit for specific histone methylation. With the device BBa_K1150024, consisting of dCas9 and G9a, we were able to decrease the endogenous VEGF expression in HEK293T cells about 50%, depending on the locus targeted (Figure XXXXXX).
Chromatin remodeling, resulting in repression of endogenous genes, is possible by fusing the histone methyltransferase G9a to dCas9.
dCas9-G9a is our most efficient repressiv device!
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| Figure X: Endogenous, stable repression by dCas9-G9a HEK293T cells have been trancfected with the BBa_K1150024 device and the RNAimer plasmid containing the different crRNA target sites for the endogenous VEGF locus. 12 hours after transfection the medium was change and 24 hours after medium change we harvested the supernatant and performed VEGF measurments by ELISA. As a control that the repressive effect of our proteins is not based on the sterical block of the transcription, we tested against the catalytic inactive dCas9. So every detectable effect is due to the G9a targeted to this locus.(n=3, p<0.05 is marked by asterisks) |
No. 3: BBa_K1150034 - uniCAS RNAimer
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| Figure XXXXX: RNAimer (BBa_K1150034) |
As dCas9 requires special RNAs for binding to the DNA, we designed a RNA plasmid containing the structure giving tracrRNA and the DNA binding crRNA. The crRNA is responsible for sequence specific DNA-binding of the entire RNA-protein complex.
The crRNA can be easily introduced to the plasmid by digesting the backbone with BbsI and insertation of an double stranded oligo. Two of these RNA plasmids (with different crRNAs) can be fused using the iGEM biobrick system. This way it is possible to get two or more crRNAs on one plasmid.
We could show that a RNAimer plasmid containing two crRNAs is as effective as co-transfected crRNAs (Figure XXXX). Besides the RNAimer enables multiple targeting, which means to use several crRNAs for one requested locus. Even for multiple targeting different RNAimers can be combined using the iGEM BioBrick system. And as the results show, multiple targeting is more effective concerning the influence the effector has on gene expression (Figure XXXXX).
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| Figure XXXXXX: RNAimer in comparison to two RNA plasmids HEK293T cells were transfected with either two RNA palsimd (left) or one RNAimer plasmid (right), containing the same crRNAs (T3, T4). The suppernant was taken and SEAP activity measured. The bars represent the mean of biological triplicates with standard deviation. |
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| Figure 7: RNAimer in comparison to two RNA plasmids SEAP activity was divided through luminescence intensity of Renilla luciferase. The bars represent the mean of biological triplicates with standard deviation. All samples were transfected with CMV:dCas9-VP16 in pSB1C3 and with two (left) or one RNA plasmid (right), each time coding for the same crRNAs. T3+4: RNAimer with T3 and T4 crRNA; T3 & T4 two different RNA plasmids with T3 and T4 crRNA. |
