Team:Alberta/Project/assemblyoverviewold

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     <h1>Overview</h1>
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     <h1>The BioBytes Alternative </h1>
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<h2>Initial Strategy</h2>
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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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<p>We are developing a streptavidin based system for the rapid and flexible assembly of great lengths of DNA. To serve as an anchor for the growing DNA chain, biotinylated single-stranded molecules of DNA are bound to streptavidin-coated paramagnetic beads. These beads allow a rapid centrifugation-free method for the exchange of wash/DNA solutions. Once the biotinylated DNA molecules are bound to the streptavidin-coated beads, a 17 base universal initiator is annealed to the single-stranded anchor. This creates a 4 base overhang that is compatible with a Not I digested DNA fragment. See <B>Figure 1A</B>. </P>
 
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<p><strong>Figure 1.</strong></p>
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<img src="https://static.igem.org/mediawiki/2009/a/a6/UofA_BBAlt_Figure1.png">
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<p><B>Figure 1A:</B> Diagrammatic representation of biotinylated anchor bound to streptavidin as well as initiator region and subsequently added bricks<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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<p>The 1st DNA “brick” to be added to the growing chain is cultivated from standard plasmids (either pAB or pBA) by restriction digest with Not I, Nb.BbvCI and Nb.BtsI. This yields a fragment with an overhang compatible with that of the initiator. The fragment is thus annealed and ligated to the initiator overhang. The other terminus of the DNA brick is comprised of a 12 base overhang known as either “A” (if taken from pBA) or “B” (if taken from pAB). See <B>Figure 1B</B>.</P>
 
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<p><strong>Figure 2.</strong></p>
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<img src="https://static.igem.org/mediawiki/2009/7/73/Bead_Figure_1BR.png">
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<img src="https://static.igem.org/mediawiki/2009/0/01/UofA_BBAlt_Figure2.png">
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<p><B>Figure 1B</B>: pAB and pBA multiple cloning sites with highlighted Not I and Nb.BbvCI/Nb.BtsI overhangs<p>
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<p>More bricks are added to the tethered chain after their removal from standard plasmids pAB and pBA. Removal of a DNA brick from pAB requires restriction digest using BspQI, Nb.BbvCI and Nb.BtsI. This yields a brick with a 12 base A overhang 5’ of the brick cassette and a 12 base B overhang 3’ of the brick cassette. See <B>Figure 2A</B>.</P>
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<br><br><br>
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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>
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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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<p><strong>Figure 3 (a).</strong></p>
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<img src="https://static.igem.org/mediawiki/2009/6/63/Bead_Figure_2A.png">
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<img src="https://static.igem.org/mediawiki/2009/a/a3/UofA_BBAlt_Figure3a.png">
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<p><B>Figure 2A</B>: pAB multiple cloning sites with highlighted A and B 12 base overhangs<p>
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<p>Removal of a DNA brick from pBA requires restriction digest using BsmBI, Nb.BbvCI and Nb.BtsI. This yields a brick with a 12 base B overhang 5’ of the brick cassette and a 12 base A overhang 3’ of the brick cassette. See <B>Figure 2B</B>. AB and BA bricks may also be PCR'd up using universal primers prA1/prB1 and prA2/prB2 respectively. This reduces the enzymes required to create the 12 base overhang to Nb.BbvCI and Nb.BtsI. PCR is the preferred method as it excludes the use of BspQI and BsmBI (which function optimally at 55/50 deg. Celsius). Regardless of creation method, these bricks are assembled sequentially onto tethered fragment via annealing of 12bp A and B regions followed by ligation.</p>
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<p><strong>Figure 3 (b).</strong></p>
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<center><img src="https://static.igem.org/mediawiki/2009/6/6d/UofA09_Bead_overviewtubular_copy.png">
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<br><br><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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<p><strong>Figure 4.</strong></p>
<center>
<center>
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<img src="https://static.igem.org/mediawiki/2009/4/42/Bead_Figure_2B.png">
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<img src="https://static.igem.org/mediawiki/2009/4/46/UofA_BBAlt_Figure4.png">
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<p><B>Figure 2B</B>: pBA multiple cloning sites with highlighted B and A 12 base overhangs<p>
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<br><br><br>
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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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<p><strong>Figure 5.</strong></p>
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<p>The tethered chain of DNA is terminated by addition of a cap (either complementary to the A overhang or the B overhang). The ligation of the Cap to the A or B overhang creates a sticky end compatible with Not I digested DNA. The construct is digested with Not I. This releases the 1st brick from the initiator/biotinylated DNA complex while creating a sticky end that will anneal to that of the Cap. The circularized DNA is then ligated a final time and transformed into E. coli using standard techniques. See <B>Figure 3</B>.</P>
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<img src="https://static.igem.org/mediawiki/2009/8/88/UofA_BBAlt_Figure5.png">
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<br><br><br>
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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>
<br>
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<p><strong>Figure 6.</strong></p>
<center>
<center>
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<img src="https://static.igem.org/mediawiki/2009/2/2a/Bead_Figure_3.png">
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<img src="https://static.igem.org/mediawiki/2009/6/6c/UofA_BBAlt_Figure6edit.png">
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<p><B>Figure 3</B>: Ligated terminators Cap (A) and Cap (B) annealed to Not I overhang created by digestion. DNA fragment is then ligated to recircularize before transformation into <i>E. coli</i><p>
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</center>
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<br>
 
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<h2>Current Strategy</h2>
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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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<h3>Brick Creation</h3>
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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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<p>The use uracil-containing primers and USER(TM) enzyme mix provides an alternative (and more effective) method for creating 12 base sticky ends. The primers anneal to the A and B regions respectively, as well as ~5bp 3' into the cassette (to increase melting temperature). Bricks cloned into pAB and pBA can be PCR'd up with these universal uracil primers prA1u/prB1u, prA2u/prB2u) and treated with USER(TM) mix. The uracil DNA glycosylase (UDG) present will cleave the uracil base and endonuclease VIII will subsequently cleave the sugar-phosphate backbone at the apyrimidinic, creating single stranded regions which can be purified away using PCR purification spin columns. See <B>Figure 4</B>. </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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<br>
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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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<img src="https://static.igem.org/mediawiki/2009/e/e0/UofA09_Bead_Overview_anchor2.png">
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<p><B>Figure 4</B>: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions<p>
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<h3>Anchoring System</h3>
 
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<p>The new and improved method for brick production (ie: USER<sup>TM</sup>) necessitated a change in anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER<sup>TM</sup>-based anchoring system. An anchoring piece, constructed of two annealed oligomers, is bound to the streptavidin-coated bead via a 5' biotin modification and provides a sticky 3' overhang complementary to an A end. When the desired number of bricks is added, a terminator (again, two annealed oligomers) is annealed and ligated to the available end of the final brick (in this case, a B end). The entire construct is then treated with USER<sup>TM</sup> enzyme mix. The resulting end, product from the digestion of uracil contained within the anchor, anneals to the terminator overhang and can be ligated to form a circular product. The ligation also yields a complete SceI site that can be used to linearize the construct for recombination into the <i>E. coli</i> genome. See <B>Figure 5</B>.</P>
 
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<img src="https://static.igem.org/mediawiki/2009/c/c0/UofA09_Bead_Overview_prA12B12u.png">
 
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<p><B>Figure 5:</B> Showing anchor and terminator fragments and effect of USER<sup>TM</sup> treatment. I SceI site and A ends are highlighted<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.