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COMBINATORIAL GIBSON DNA ASSEMBLY
COMBINATORIAL GIBSON DNA ASSEMBLY
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Written by Daniela Alvarez
Updated over a week ago

Modern industrial cloning techniques have made the construction of very large stretches of DNA much easier. In addition, the simple "single pot" nature of the reactions for assembling multiple fragments has allowed researchers to build many combinatorial variants straightforwardly. A recent Cold Spring Harbor book has some good background information if you want to dig into the details.

By way of introduction to the method that broke open the field, let's do a simple combinatorial design using the Gibson method which relies on flanking homology to enable scarless DNA assembly.

To start a new design, click on the “Designs” option on the Top Menu of the Design Module and select “Designs”.

Right after, you will see the typical library view with the designs you have worked on; to create a new Combinatorial Gibson Assembly click on the “New Design” option on the upper part of the library, then select “Load Example Design” and select the Assembly method desired, (on this case “Gibson”). After that, you will be asked to select a name for your design.

The example Combinatorial Gibson DNA Assembly will show 7 different parts. Each one has a representative icon on the first row and the name of the part on the second one.

If you click on one of the icons, a right-side panel will appear, showing you the Bin Details of that icon.

On this panel, you can change the icon by clicking on the image to display the list of available SBOL icons. You can also change the name, indicate if it’s a direct synthesis firewall, direction, forced relative overhang position, extra 5’ and 3’ overlap Bps, the list of the parts included in that icon, and edit and validate new design rulesets.

In the same way, this panel will appear if you click on the name of a part (second row), showing you the Part Details.

There, you can go and view all the part details, update tags (by clicking on the icon), see the name of the part, part source and its details (size and reverse complement), sequence properties, and digest part. On the bottom part of the panel, you can add notes on the part and indicate your Preferred 5' and 3’ Overhangs, as well as the Forced Assembly Strategy and Eugene rules.

The available Forced Assembly Strategies are:

  • None

  • Digest

  • Direct Synthesis

  • PCR

  • Embed in primer forward

  • Embed in primer reverse

  • Annealed Oligos

  • Top Strand Oligo

  • Assembly Ready Fragment

Notice that, if you click on “View Part” you will see a new screen showing you the typical DNA Sequence view.

Similarly, clicking on “Part Source” takes you to the DNA Sequences library, where you can change the details of your sequence.

Going back to the Design screen, by double-clicking on the name of a part, a window will be displayed showing the part details. This window allows you to change the part name, indicate if the strand is positive or negative, and indicate the start and end positions. On the mright corner, you can also show/hide the features and parts, and change to a linear or sequence view.

Notice that there is a right menu on your screen, shown as a blue right panel with icons. The first option corresponds to the information icon, that shows you your Design Details. There, you can change the name and description of your design, select between combinatorial/list Layout Types, indicate whether you want to reuse oligos from the library and remove interrupted features, export to JSON format, and create a schematic from this design.

The second option on the right panel shows you the list of the Assembly Reports.

The third option shows and allows you to edit the Assembly Reaction Details, including the number of possible combinations, presets, and assembly method and parameters. You can also customize the name given to the assembled constructs, oligo synthesis, DNA synthesis, PCR reactions, and assembly pieces. Notice that the number of possible combinations is an upper bound, which means it will ignore the Eugene rules.

The fourth option “Card Details”

There you can also see the construct annotations. This creates parts or features on the resulting assembled constructs. To create a new one, click on the “new” icon and fill in the required information.

The next two sections of the menu are Bin Details and Part Details, the same ones that can be accessed by clicking on the icons or names of the parts respectively. The following option, Junction Details

Right after, the Design Comments option allows you to see past comments, reply to them, and create new ones.

The last option, Reviews, creates a review request for the Lab Manager. If you are not logged in on an admin account, you can only ask for a review and add a comment.

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