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Team:Alberta/biobyte2
From 2010.igem.org
Contents |
Components of the System
The Assembly Method 2.0 is composed of three main components. An anchor byte attached to an iron micro bead is the beginning of a construct. Because of the magnetic nature of these beads, they can be positioned by using a simple magnet. The BioBytes are added to the anchor-byte one at a time in sequence. This is possible due to the alternating overhang structure of the BioBytesa. Finally, a cap is added allowing for circularization of the construct. The construct is now ready to transform
Overhangs
Anchor Byte
The Anchor-byte begins the process of assembly. It is composed of:
- a poly-A tail
- a BsaI recognition site
- an A or B overhang
Iron micro beads purchased from New England Biolabs have covalently attached poly-T tails. The bead allows us to manipulate the DNA with magnets making washing and subsequent attachments easier.
We have designed an anchor byte which begins the process of assembly. The anchor-byte is comprised of an Anchor piece ligated to the first byte of the assembly. Construction begins by ligating a selectable marker to the anchor. This first step allows for complete constructs to be selected for. As well, the incorporation of a BsaI cut site into the Anchor, before the first byte, gives versatility to the construct because the first byte and the rest of the construct can be removed from the anchor, and used as a Byte in and of itself.
Once the selectable marker is ligated to the first byte, we anneal the anchor-byte to the poly-T tails on the iron micro bead. We have created anchors with both varieties of ends so that assemblies can begin with any type of byte. The results of one of our experiments is shown here. Note that the interaction between the anchor and the bead is non-covalent. The anchor along with the construct can be separated from the bead with heat. The lefthand gel shows the process of anchoring a construct. A anchor-byte construct of 1kb is allowed to anneal to the magnetic beads. This is done in excess, and the supernatant is shown in the first lane. A subsequent wash step showed the absence of DNA, indicating that DNA construct is stably bound. The construct can be melted from the scaffold at 70 degrees Celsius. The melted construct can be seen in the last lane.
This schematic shows the alternating addition of bytes starting from the anchor. Parts are added and ligated in an sequential fashion. Cycle time for each step was about 7 minutes. This is much faster than the Biobrick method. In on of our assemblies, we were able to create an octamer, with a total size of 12 kb. As you can see, there are some minor incomplete products.
If you recall, we have incorporated a BsaI cut site into the anchor. This allows for constructs to be created in parallel and then utilized as large Bytes in the assembly in the same way.
