Team:Botchan Lab Tokyo/Design

Botchan Lab. Tokyo

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Design

Design

Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.

What should this page contain?

• Explanation of the engineering principles your team used in your design
• Discussion of the design iterations your team went through
• Experimental plan to test your designs

Inspiration

• 2016 MIT
• 2016 BostonU
• 2016 NCTU Formosa

Index

• Points of Attention in Design
• The Best Method to Achieve Our Purpose
• Outline our design
• Reference

Points of Attention in Design

In our project, we need to introduce three types of radioresistance genes in the end. Also, the gene we want to introduce is not an operon. Furthermore, each gene does not form a cluster. It is scattered in chromosome1 and plasmid of Deinococcus Radiodurans. In addition to these, there were some things we had to be careful about when making parts. Therefore, we paid attention to the following points when designing the parts.

• Even if there are multiple genes to be introduced, it must be cheap and easy to introduce in a short time.
• There must be one promoter at the beginning of each gene. In other words, when we insert multiple genes, promoters should be introduced according to the number of inserts.
• Adjust the joints between genes and prefix, suffix to create a restriction enzyme site to make the parts compatible with biobrick.
• PCR to add his tag is necessary.

The Best Method to Achieve Our Purpose So what's the best way to meet these conditions? Our answer was to take the in-fusion cloning method.

In-fusion cloning

In-fusion cloning is a method that inserts genes by adding homologous sequences and connecting them by using a kit, not by restriction enzyme treatment. It has following characteristics.

• We can clone any insert, into any location, within any vector we choose.
• We can clone multiple DNA fragments simultaneously into any vector in a single reaction.
• No need for restriction digestion, phosphatase treatment, or ligation.
• Final constructs are seamless with no extra or unwanted base pairs.
• The kit is cheap.
• We can add his tag by inverse PCR which is in the procedure.

In a little more detail, In-fusion cloning can be roughly divided into 8 steps.

• 1st step: Linearization of vector (inverse PCR)
• 2nd step: Making primers to create transgenes with homologous sequences to vectors
• 3rd step: Amplification of target gene
• 4th step: Making target gene with homologous sequence added
• 5th step: Incubate PCR products with cloning enhancer or purify on spin column
• 6th step: Set up cloning
• 7th step: Proceeding the cloning reaction.
• 8th step: Transform competent cells.

If we use in-fusion cloning with these characteristics, we can solve the problems we mentioned above.

Outline our design

We decided to introduce RecA, PqqE, and PprM genes from Deinococcus radiodurans by using in-fusion cloning.

RecA

Without His tag

We added a homologous sequence with psB1C3 to RecA we took by PCR from Deinococcus radiodurans during PCR.Similarly, we added a homologous sequence of psB1C3 and RecA to the sequence between the fully synthesized prefix and RecA which include the promoter and RBS within them by PCR, then we proceeded in-fusion cloning.

With His tag

We added a homologous sequence with psB1C3 to RecA we took by PCR from Deinococcus radiodurans during PCR, and proceeded in-fusion cloning to get plasmid only with RecA in it.

Next, add his tags to the 5' end of RecA during inverse PCR, and similarly, we added a homologous sequence of psB1C3 and RecA that has his tag at the beginning, to the sequence between the fully synthesized prefix and RecA which include the promoter and RBS within them by PCR, then we proceeded in-fusion cloning.

PqqE

Without His tag

We add a homologous sequence with psB1C3 to PqqE we take by PCR from Deinococcus radiodurans during PCR. Similarly, we add a homologous sequence of psB1C3 and PqqE to the sequence between the fully synthesized prefix and PqqE which include the promoter and RBS within them by PCR, then we proceed in-fusion cloning.

>With His tag

We add a homologous sequence of psB1C3 to PqqE from Deinococcus radiodurans that has his tag we add, by PCR. Then we proceed in-fusion cloning to get plasmid only with PqqE in it.

Next, similarly, we add a homologous sequence of psB1C3 and PqqE to the sequence between

the fully synthesized prefix and PqqE which include the promoter and RBS within them by PCR, then we proceed in-fusion cloning.

PprM

Without His tag

We add a homo logous sequence with psB1C3 to PprM we take by PCR from Deinococcus radiodurans during PCR.

Similarly, we add a homologous sequence of psB1C3 and PprM to the sequence between the fully synthesized prefix and PprM which include the promoter and RBS within them by PCR, then we proceed in-fusion cloning.

With His tag

We add a homologous sequence of psB1C3 to PprM from Deinococcus radiodurans that has his tag we add, by PCR. Then we proceed in-fusion cloning to get plasmid only with PprM in it.

Next, similarly, we add a homologous sequence of psB1C3 and PprM to the sequence between the fully synthesized prefix and PprM which include the promoter and RBS within them by PCR, then we proceed in-fusion cloning.

RecA+PprM

Without His tag

We insert the sequence from the prefix to the terminator that has RecA in it into a plasmid which only has PprM in it by in-fusion cloning.

With His tag

We insert the sequence from the prefix to the terminator that has RecA which has his tag on

its 5’ end into a plasmid which only has PprM in it by in-fusion cloning.

PqqE+PprM

Without His tag

We insert the sequence from the prefix to the terminator that has PqqE in it into a plasmid which only has PprM in it by in-fusion cloning.

With His tag

We insert the sequence from the prefix to the terminator that has PqqE which has his tag into a plasmid that only has PprM in it by in-fusion cloning.

PqqE+RecA

Without His tag

We insert the sequence from the prefix to the terminator that has PqqE in it into a plasmid which only has RecA in it by in-fusion cloning.

With His tag

We insert the sequence from the prefix to the terminator that has PqqE which has his tag into a plasmid that only has RecA in it by in-fusion cloning.

PqqE+PprM+RecA

Without His tag

We insert the sequence from the prefix to the terminator that has RecA in it into a plasmid which has PqqE and PprM in it by in-fusion cloning.

With His tag

We insert the sequence from the prefix to the terminator that has RecA which has his tag on its 5’ end into a plasmid which has PqqE and PprM in it by in-fusion cloning.

Reference

• In-Fusion®HD Cloning Kit User Manual., Takara Bio USA, Inc., p.3-4.

Botchan lab. Tokyo

Botchan lab. Tokyo