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The redoxilator
- Genetic design
- Applications and perspectives
- Safety considerations
The USER assembly standard
- USER fusion of biobricks
USER fusion primer design software
- Abstract
- Instructions
- Output format
Experimental results
- Results and discussion
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The project
The USER fusion assembly standard
The USER fusion assembly standard allows rapid construction of multi-part devices, without some of the drawbacks of the restriction-enzyme based standard biobrick assembly method. The full USER assembly standard can be found here: (BBF RFC 39). The main advantages of this assembly method is:
1. Standardized method for assembling several BioBricks or components at a time, in contrast to the one at a time" assembly procedure normally used.
2. Since the method relies PCR based assembly, all restriction sites are allowed in the biobricks.
3. Eight basepair-overhangs allows ligase-free cloning. With the enclosed protocol E. coli can be transformed with a multipart-construct less than 2 hours after your PCR-reaction has completed.
4. The biobricks are joined without leaving a scar which is ideal for fusing protein domain biobricks.
5. Insertions of small sequences between biobricks such as a intracellular localization signal, restriction site or flexible linker is possible with the right primer design.
6. High fidelity is ensured by using PfuTurbo® Cx Hotstart DNA polymerase.
7. By the design of the PCR tails, it can be decided whether the USER cassette should be deleted, copied or moved following insertion.
8. Directionality of inserts are supported.
USER fusion of biobricks - how it works
We have successfully constructed a USER fusion biobrick (BBa_K194003), and demonstrated that it works as expected. The biobrick includes a DNA-sequence needed for USER-fusion, which consists of a restriction site and two nicking sites. The entire process of fusing two biobricks are illustrated in the figure, and the same can be done for multiple fragments at once.
Procedure (please refer to our Biobrick Assembly Standard BBF RFC 39 for a detailed protocol):
1. The USER fusion biobrick plasmid is digested with the restriction enzyme pacI and the nicking enzyme Nt.BbvCI (a nicking enzyme cuts only one strand as illustrated on the figure). This process will linearize the plasmid, and make single stranded overhangs (sticky ends).
2. PCR amplification is performed on the biobricks intended for the fusion. The primer design is facilitated by our novel USER fusion primer design software made for this iGEM project.
3. USER enzyme mix is added. This will remove the uracil always included in the primers, making sticky end overhangs on all biobricks. Because of the matching sticky ends on all biobricks and linearized plamid, the biobricks will self-assemble in the plasmid.
Two or more biobricks have been joined with all the advantages mentioned above.
Design your primers with PHUSER here
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Achievements
Redox sensing device
Two novel genes have been designed and synthesized each comprised of 5 genetic elements. Together they function as a device termed the Redoxilator that can sense the internal redox state of a yeast cell, and output a reporter signal. Extensive mathematical modelling was performed to simulate how the construct would operate in vivo.
Biobricks
DNA of several new biobricks have been designed and submitted including a yeast optimized GFP reporter protein, a protein degradation sequence and a fast degradable yeast GFP. (Bronze medal)
We have demonstrated that our USER fusion biobrick works as expected and documented it (silver medal)
USER fusion Assembly standard
A new biobrick assembly standard that allows the rapid construction of multi-part devices have been developed and documented. The assembly standard offers many benefits: All restriction sites are allowed, multiple biobricks can be joined in one step, the result is scar-free making it ideal for protein fusions and more. (Gold medal)
USER-fusion primer design software
A novel and very useful software tool have been developed that can automatically design the optimal primers for USER fusion assembly of 2-9 biobricks, taking several parameters into account.
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