Team:Berkeley Wetlab/Automation

Why Automate?

Our approach to characterizing cell surface display required us to make a lot of parts. Without an automated mode of assembly it would not be possible for us to make enough parts to characterize cell surface display using a combinatorial method.

Making Basic Parts

Making basic parts is the first step toward making any synthetic biology device. After designing all of the basic parts we needed for our cell surface display project, our team developed a method for doing cheap in-house gene synthesis. This method allowed us to easily make a very large number of basic parts.

oligoDesigner

We created a software called oligoDesigner to facilitate gene synthesis. OligoDesigner will take a list of goal part sequences as input and output all of the oligo nucleotides needed to synthesize the goal part. OligoDesigner will also output an IDT ([http://www.idtdna.com/Home/Home.aspx Integrated DNA Technologies]) order form that will order all of the oligo nucleotides it designed.

oligoDesigner

IDT Ordering Form

(IDT 96 well plate image)

Liquid Handling Robot

Basic Parts

The oligo nucleotides arrive from IDT in a 96 well plate format. OligoDesigner will then generate commands for a liquid handling robot to resuspend the oligo nucleotides to the correct concentrations. It will also generate commands that tell the liquid handling robot how to mix the oligos in order to get the correct PCR products.
After the liquid handling robot finishes mixing oligo nucleotides, all we need to do to get our basic parts is add the correct buffers and enzymes and put the resultant tubes in a thermocycler for PCR.

Making Composite Parts

The next step, after making new basic parts, is to assemble those basic parts into composite part devices. There has been a lot of work being done at UC Berkeley to automate the process of composite part assembly. This year our team was able to implement many newly developed techniques to create a large number of composite parts.

2AB Layered Assembly

Our team used a method of basic part assembly called Two AntiBiotic (2AB) Assembly to create composite parts. 2AB assembly is a straight-forward and robust method for combining two or more basic parts into a destination vector. It is a layered assembly scheme used by the Anderson Lab at UC Berkeley. For more information about 2AB Assembly please visit the Layered Assembly page of the 2008 Berkeley iGEM wiki.

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The 2AB assembly method lends itself well to automation because it consists mostly of liquid handling. Basic parts must be put into the correct specialized assembly vector, and then mixed with other basic parts in the correct order, to yield goal composite parts. The process of putting basic parts into the correct specialized assembly vectors is called "gateway". An in vivo gateway scheme amenable to high throughput was developed by the 2008 Berkeley iGEM team. This year we were able to use their in vivo gateway scheme to put our basic parts into the correct specialized assembly vectors. To read more about in vivo gateway visit the 2008 Berkeley iGEM wiki here.

Clotho and Liquid Handling Robot

2AB assembly utilizes six different types of specialized assembly vectors

• liquid handling robots

Success!

Using our newly developed automated assembly processes we were able to make over 800 parts (both basic and composite) in one summer! This magnitude of part making is unprecedented in iGEM competitions and enabled us to test a very large number of variations on cell surface display systems.

Success Rates

The assembly scheme we developed works just as well as assembly by hand and is much faster.

• percentages
• see Jenn Brophy and Gabriela Guzman's notebooks for notes on assembly.
• 2ab assembly
• Clotho
• statistics

We made a lot of parts with a robot.

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