Team:Alberta/Project/assemblyoverviewold

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     <h1>Overview of DNA Assembly</h1>
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     <h1>The BioBytes Alternative </h1>
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<p>BioBytes constitutes the University of Alberta’s contribution to iGEM 2009. It is a next-generation gene assembly system with the singular potential to accelerate the field toward the grand vision of the artificial cell. Unlike BioBricks, genes produced in the BioByte format can be assembled rapidly in vitro, in any desired order, with great precision and yield. With cycle times approaching 20 minutes for the addition of each new gene, BioByte assembly rates exceed their BioBrick counterparts by 200-fold. This level of improvement immediately opens the door towards the synthesis of simple chromosomes that can be tested and optimized at unprecedented speed. Finally, the BioByte assembly system requires a fraction of the equipment found in a conventional gene lab. This advantage, combined with a "child-could-do-it" simplicity, greatly extends its utility from the high school classroom to the workbench of the bio-process engineer. </p>
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<p>A BioByte is a piece of DNA that encodes a specific type of cellular function or instruction. Each end of the DNA is distinct from the other so that they can only be joined in a head-to-tail fashion as shown below. </p>
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<p align=right>
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<a href="https://2009.igem.org/Team:Alberta/ByteCreation">Click here for more on byte creation...</a></p>
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<br>
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<p><strong>Figure 1.</strong></p>
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<br><br><br>
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<p>Once correctly paired, Bytes are irreversibly linked together by an enzyme called DNA ligase. It is the strength and accuracy of the head-to-tail interaction that accounts for BioByte’s superior precision and speed relative to its BioBrick predecessor. </p>
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<p>The hand-in-glove nature of the BioByte interaction illustrates not only how the orientation of each Byte is determined, but also how this approach can be used to control the order by which Bytes are assembled. </p>
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<br>
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<p><strong>Figure 2.</strong></p>
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<p>The problem of determining Byte order is shown in Figure 2. Here, the free ends on the existing two-byte chain are such that the incoming third brick can be added randomly to either end, resulting in orders like [1-2-3] or [3-1-2]. BioBytes has overcome this problem by using a third type of end that can only be linked to an inert magnetic microsphere (Figure 3a). By anchoring the first byte to the microsphere via this new end, only one end (its free end) is available for interaction. The final anchoring system was chosen after exploring several avenues for anchor design.
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<p align=right><a href="https://2009.igem.org/Team:Alberta/DNAanchor">Click here for more on anchor selection...</a></P><P>
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The chain is therefore constrained to grow in only one direction: away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. A magnet can be applied to the side of an Eppendorf tube to gather the beads and allow for convenient solution change. In this way, a solution containing a Byte can be applied to the beads, ligated, and have the unlinked Bytes removed by wash steps rapidly. The subsequent Byte is then added and the process begins again (Figure 3b).</p>
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<br>
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<p><strong>Figure 3 (a).</strong></p>
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<img src="https://static.igem.org/mediawiki/2009/a/a3/UofA_BBAlt_Figure3a.png">
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<p><strong>Figure 3 (b).</strong></p>
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<BR>
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<p>At this stage, the problem of Byte order and chain fidelity is not entirely solved. The method described above cannot exclude the possibility that multiple copies of a particular Byte become incorporated at a given step, as shown in Figure 4 below. </p>
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<br>
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<p><strong>Figure 4.</strong></p>
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<p>The problem arises because each Byte that is incorporated into the chain does not enter the reaction mixture as a single molecule, but as a population of identical molecules that are as likely to interact with each other as the anchored chain (Figure 5). </p>
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<br>
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<p><strong>Figure 5.</strong></p>
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<p>BioBytes solves the problem by constructing each Byte in two alternative forms: an “AB” form and a “BA” form. Each form has two incompatible ends. Neither form can be linked to itself (Figure 6). However, the ends of each form are compatible with each other, allowing for the alternating order of AB and BA forms in a head-to-tail orientation. Adding Bytes to the growing chain by alternating the AB an BA forms assures that only one copy of each is added at each step. </p>
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<br>
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<p><strong>Figure 6.</strong></p>
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<img src="https://static.igem.org/mediawiki/2009/6/6c/UofA_BBAlt_Figure6edit.png">
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<p>Upon completion of the desired product, the chains can be released from the microspheres by a chemical cleavage event to yield the desired linear construct.
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</P>
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<p align=right><a href="https://2009.igem.org/Team:Alberta/Project/linearconstruct">Click here for results in linear construction...</a></P><P>
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Alternatively, an annealed terminator may be added to the chain. This allows recircularization upon release of the construct from the microspheres. The recircularized constructs can then be introduced into living cells and propagated, provided an origin of replication was included during construction.</P>
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</P>
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<p align=right><a href="https://2009.igem.org/Team:Alberta/plasmidconstruct">Click here for results in plasmid construction...</a></P><P>
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<p>With its BioBytes approach, the Alberta team has recently demonstrated the accurate construction of chains composed of 10 Bytes over the course of 4 hours with no obvious limit to the final length that can be achieved. A key advantage to this approach is that it can be multiplexed for the production of multiple chains simultaneously that can be linked together as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.</p>
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<p> The protocol for BioByte assembly can be found <a href="https://2009.igem.org/Team:Alberta/Project/ByteAssembly">here</a>.
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Latest revision as of 22:55, 21 October 2009

University of Alberta - BioBytes










































































































The BioBytes Alternative

BioBytes constitutes the University of Alberta’s contribution to iGEM 2009. It is a next-generation gene assembly system with the singular potential to accelerate the field toward the grand vision of the artificial cell. Unlike BioBricks, genes produced in the BioByte format can be assembled rapidly in vitro, in any desired order, with great precision and yield. With cycle times approaching 20 minutes for the addition of each new gene, BioByte assembly rates exceed their BioBrick counterparts by 200-fold. This level of improvement immediately opens the door towards the synthesis of simple chromosomes that can be tested and optimized at unprecedented speed. Finally, the BioByte assembly system requires a fraction of the equipment found in a conventional gene lab. This advantage, combined with a "child-could-do-it" simplicity, greatly extends its utility from the high school classroom to the workbench of the bio-process engineer.

A BioByte is a piece of DNA that encodes a specific type of cellular function or instruction. Each end of the DNA is distinct from the other so that they can only be joined in a head-to-tail fashion as shown below.

Click here for more on byte creation...


Figure 1.




Once correctly paired, Bytes are irreversibly linked together by an enzyme called DNA ligase. It is the strength and accuracy of the head-to-tail interaction that accounts for BioByte’s superior precision and speed relative to its BioBrick predecessor.

The hand-in-glove nature of the BioByte interaction illustrates not only how the orientation of each Byte is determined, but also how this approach can be used to control the order by which Bytes are assembled.


Figure 2.




The problem of determining Byte order is shown in Figure 2. Here, the free ends on the existing two-byte chain are such that the incoming third brick can be added randomly to either end, resulting in orders like [1-2-3] or [3-1-2]. BioBytes has overcome this problem by using a third type of end that can only be linked to an inert magnetic microsphere (Figure 3a). By anchoring the first byte to the microsphere via this new end, only one end (its free end) is available for interaction. The final anchoring system was chosen after exploring several avenues for anchor design.

Click here for more on anchor selection...

The chain is therefore constrained to grow in only one direction: away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. A magnet can be applied to the side of an Eppendorf tube to gather the beads and allow for convenient solution change. In this way, a solution containing a Byte can be applied to the beads, ligated, and have the unlinked Bytes removed by wash steps rapidly. The subsequent Byte is then added and the process begins again (Figure 3b).


Figure 3 (a).

Figure 3 (b).





At this stage, the problem of Byte order and chain fidelity is not entirely solved. The method described above cannot exclude the possibility that multiple copies of a particular Byte become incorporated at a given step, as shown in Figure 4 below.


Figure 4.




The problem arises because each Byte that is incorporated into the chain does not enter the reaction mixture as a single molecule, but as a population of identical molecules that are as likely to interact with each other as the anchored chain (Figure 5).


Figure 5.




BioBytes solves the problem by constructing each Byte in two alternative forms: an “AB” form and a “BA” form. Each form has two incompatible ends. Neither form can be linked to itself (Figure 6). However, the ends of each form are compatible with each other, allowing for the alternating order of AB and BA forms in a head-to-tail orientation. Adding Bytes to the growing chain by alternating the AB an BA forms assures that only one copy of each is added at each step.


Figure 6.

Upon completion of the desired product, the chains can be released from the microspheres by a chemical cleavage event to yield the desired linear construct.

Click here for results in linear construction...

Alternatively, an annealed terminator may be added to the chain. This allows recircularization upon release of the construct from the microspheres. The recircularized constructs can then be introduced into living cells and propagated, provided an origin of replication was included during construction.

Click here for results in plasmid construction...

With its BioBytes approach, the Alberta team has recently demonstrated the accurate construction of chains composed of 10 Bytes over the course of 4 hours with no obvious limit to the final length that can be achieved. A key advantage to this approach is that it can be multiplexed for the production of multiple chains simultaneously that can be linked together as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.

The protocol for BioByte assembly can be found here.