Team:Alberta

From 2009.igem.org

(Difference between revisions)
Line 51: Line 51:
<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 in vitro rapidly, in any desired order, at great precision and yield. With cycle times approaching 15 minutes for the addition of each new gene, BioByte assembly rates exceed their biobrick counterpart 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>
<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 in vitro rapidly, in any desired order, at great precision and yield. With cycle times approaching 15 minutes for the addition of each new gene, BioByte assembly rates exceed their biobrick counterpart 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>
<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 so that they can only be joined in a head-to-tail fashion as shown below. </p>
<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 so that they can only be joined in a head-to-tail fashion as shown below. </p>
 +
<br>
<p><strong>Figure 1.</strong></p>
<p><strong>Figure 1.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/a/a6/UofA_BBAlt_Figure1.png">
<img src="https://static.igem.org/mediawiki/2009/a/a6/UofA_BBAlt_Figure1.png">
 +
<br><br><br>
<p>Once correctly paired, Bytes are irreversibly linked together by a special enzyme called DNA ligase. It is the strength and accuracy of the head-to-tail interaction that accounts for BioByte’s superior yield, precision and speed relative to its BioBrick predecessor. </p>
<p>Once correctly paired, Bytes are irreversibly linked together by a special enzyme called DNA ligase. It is the strength and accuracy of the head-to-tail interaction that accounts for BioByte’s superior yield, precision and speed relative to its BioBrick predecessor. </p>
<p>The hand-in-glove nature of the BioByte interaction illustrates how the orientation of each Byte is determined, but how can this approach be used to control the order by which Bytes are assembled? </p>
<p>The hand-in-glove nature of the BioByte interaction illustrates how the orientation of each Byte is determined, but how can this approach be used to control the order by which Bytes are assembled? </p>
 +
<br>
<p><strong>Figure 2.</strong></p>
<p><strong>Figure 2.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/0/01/UofA_BBAlt_Figure2.png">
<img src="https://static.igem.org/mediawiki/2009/0/01/UofA_BBAlt_Figure2.png">
 +
<br><br><br>
<p>The problem of determining Byte order is shown in Figure 2. Here, the free ends on the existing two-byte chain means that the incoming third brick can be added randomly to either end resulting in the order [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 3). By anchoring the first byte to the microsphere via this new end, only its free end is available for interaction. The chain therefore is constrained to grow in only one direction, away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. Anchored chains can therefore be moved out of one reaction mixture leaving unlinked bytes behind, into a new reaction that containing new byte molecules needed for the next round of addition. </p>
<p>The problem of determining Byte order is shown in Figure 2. Here, the free ends on the existing two-byte chain means that the incoming third brick can be added randomly to either end resulting in the order [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 3). By anchoring the first byte to the microsphere via this new end, only its free end is available for interaction. The chain therefore is constrained to grow in only one direction, away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. Anchored chains can therefore be moved out of one reaction mixture leaving unlinked bytes behind, into a new reaction that containing new byte molecules needed for the next round of addition. </p>
 +
<br>
<p><strong>Figure 3.</strong></p>
<p><strong>Figure 3.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/a/a3/UofA_BBAlt_Figure3a.png">
<img src="https://static.igem.org/mediawiki/2009/a/a3/UofA_BBAlt_Figure3a.png">
<img src="https://static.igem.org/mediawiki/2009/9/96/UofA_BBAlt_Figure3b.png">
<img src="https://static.igem.org/mediawiki/2009/9/96/UofA_BBAlt_Figure3b.png">
 +
<br><br><br>
<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>
<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>
 +
<br>
<p><strong>Figure 4.</strong></p>
<p><strong>Figure 4.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/4/46/UofA_BBAlt_Figure4.png">
<img src="https://static.igem.org/mediawiki/2009/4/46/UofA_BBAlt_Figure4.png">
 +
<br><br><br>
<p>The problem arises because each byte that is incorporated into the chain does not enter the reaction mixture as 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>
<p>The problem arises because each byte that is incorporated into the chain does not enter the reaction mixture as 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>
 +
<br>
<p><strong>Figure 5.</strong></p>
<p><strong>Figure 5.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/8/88/UofA_BBAlt_Figure5.png">
<img src="https://static.igem.org/mediawiki/2009/8/88/UofA_BBAlt_Figure5.png">
 +
<br><br><br>
<p>BioBytes solves the problem by constructing each Byte in two alternative forms, an “A” form and a “B” form. Each form has two incompatible ends. Therefore neither form can be linked to itself (Figure 6). The ends of each form are however, are compatible to each other, allowing for the alternating order of A and B forms in head-to-tail orientation. Adding Bytes to the growing chain by alternating the A an B forms assures that only one copy of each is added at each step </p>
<p>BioBytes solves the problem by constructing each Byte in two alternative forms, an “A” form and a “B” form. Each form has two incompatible ends. Therefore neither form can be linked to itself (Figure 6). The ends of each form are however, are compatible to each other, allowing for the alternating order of A and B forms in head-to-tail orientation. Adding Bytes to the growing chain by alternating the A an B forms assures that only one copy of each is added at each step </p>
 +
<br>
<p><strong>Figure 6.</strong></p>
<p><strong>Figure 6.</strong></p>
<img src="https://static.igem.org/mediawiki/2009/7/7e/UofA_BBAlt_Figure6.png">
<img src="https://static.igem.org/mediawiki/2009/7/7e/UofA_BBAlt_Figure6.png">
 +
<br><br><br>
<p>Upon completion of the desired product, chains are released from the microspheres by a chemical cleavage event that separates the anchor brick from its bound end, and are then introduced into living cells.</p>  
<p>Upon completion of the desired product, chains are released from the microspheres by a chemical cleavage event that separates the anchor brick from its bound end, and are then introduced into living cells.</p>  
<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 many different chains simultaneously, that, can all be linked as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.</p>
<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 many different chains simultaneously, that, can all be linked as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.</p>

Revision as of 06:29, 28 September 2009

University of Alberta - BioBytes










































































































BioBytes

Team BioBytes is the University of Alberta's 2009 International Genetically Engineered Machines (iGEM) team. This year's iGEM project can be subdivided into two major efforts. The first and most important of which is the BioBytes chromosome assembly system. This system refers to a mechanism for rapid and reliable construction of plasmids (i.e.: artificial gene sets) in vitro. The second, the minimal genome project, refers to the ultimate goal of rapid and reliable DNA assembly, that is, the construction of an artificial E. coli chromosome. Furthermore, it includes the strategy of gene selection, arrangement, artificial chromosome insertion and the destruction of the host's chromosome.

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 in vitro rapidly, in any desired order, at great precision and yield. With cycle times approaching 15 minutes for the addition of each new gene, BioByte assembly rates exceed their biobrick counterpart 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 so that they can only be joined in a head-to-tail fashion as shown below.


Figure 1.




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

The hand-in-glove nature of the BioByte interaction illustrates how the orientation of each Byte is determined, but how can this approach 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 means that the incoming third brick can be added randomly to either end resulting in the order [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 3). By anchoring the first byte to the microsphere via this new end, only its free end is available for interaction. The chain therefore is constrained to grow in only one direction, away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. Anchored chains can therefore be moved out of one reaction mixture leaving unlinked bytes behind, into a new reaction that containing new byte molecules needed for the next round of addition.


Figure 3.




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 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 “A” form and a “B” form. Each form has two incompatible ends. Therefore neither form can be linked to itself (Figure 6). The ends of each form are however, are compatible to each other, allowing for the alternating order of A and B forms in head-to-tail orientation. Adding Bytes to the growing chain by alternating the A an B forms assures that only one copy of each is added at each step


Figure 6.




Upon completion of the desired product, chains are released from the microspheres by a chemical cleavage event that separates the anchor brick from its bound end, and are then introduced into living cells.

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 many different chains simultaneously, that, can all be linked as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.

The Minimal Genome Project

The minimal E. coli genome has been a holy grail of biology for a number of years. E. coli is the most widely used cellular research tool by the molecular biology community. Since scientific research is based upon reductionism and simplification for understanding, a simplified version of an experimental model organism such as E. coli is, in principle, preferred as a chassis for experimentation. To reduce the E. coli genome to roughly 10% its original size shows a great simplification of this model organism.

To create such an organism, we plan on building an artificial E. coli chromosome using the BioBytes chromosome assembly system and inserting it into living E. coli. We then intend to remove the host chromosome by making it incapable of division. This allows only the artificial, inserted chromosome to propagate through multiple generations as the cells grow and divide. This is markedly different than the current, time-consuming method of knocking out inessential genes, one at a time, in an effort to produce the minimal genome. It is this difference that we hope to exploit in our attempt to win the race to produce the minimal E. coli genome.

Get a free html hit counter here.