Team:NYU/Assembly

From 2010.igem.org

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[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]
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This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms.
 
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[[Image:NYU_Overlapassembly.jpg‎|300px|left]]
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This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. By combining your overlapping DNA parts with the required enzymes and buffers you can easily assemble a complete system in one day - perfect for the impatient New Yorkers that we are. Here's the general schematic:
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To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us -  check it out by clicking this link:
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[[Image:NYU_Overlapassembly.jpg‎|300px|center]]
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The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us -  check it out by clicking this link:
<html>
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<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="http://2010.igem.org/wiki/images/5/5c/Overlaplogo.gif"  /></a></html>
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="http://2010.igem.org/wiki/images/5/5c/Overlaplogo.gif"  /></a></html>
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After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:
+
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, Russell designed a simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this simple mix:
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50uL Assembly Reaction:
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  Equimolar amounts of overlapping DNA (~100ng) from PCR cleanup - up to 18uL
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      +
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  10uL  5x Phusion HF Polymerase Buffer
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  5uL  10x Taq Ligase Buffer
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  8uL  50mM MgCl2
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  4uL  2.5mM dNTPs
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  .5uL  ExoIII diluted to 10 U/uL
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  2uL  Phusion HF Polymerase (courtesy of Finnzyme)@ 2 U/uL
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  4uL  Taq Ligase @ 40 U/uL
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      +
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  Water to 50uL
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ENTER REACTION MIX HERE
 
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We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.
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We kept this reaction mix at 50C for 60 seconds to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C at a very slow ramp rate the now-single-stranded parts anneal to one another, allowing the Phusion polymerase and Taq ligase to fill in and closed the gaps. Because the yields are too low to clone directly from this reaction we PCRed full constructs using BB prefix and suffix primers and high fidelity polymerase which could then be cloned into a biobrick-compatible plasmid.

Revision as of 04:36, 27 October 2010



NYU OverviewSideF 3.png

While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system.

We decided to use the Overlap Assembly Method most famously published in this JCVI paper:

Gibson et al, Nature Methods 6: 343-345


This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. By combining your overlapping DNA parts with the required enzymes and buffers you can easily assemble a complete system in one day - perfect for the impatient New Yorkers that we are. Here's the general schematic:

NYU Overlapassembly.jpg


The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:

After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, Russell designed a simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this simple mix:

50uL Assembly Reaction:

  Equimolar amounts of overlapping DNA (~100ng) from PCR cleanup - up to 18uL
      +
  10uL  5x Phusion HF Polymerase Buffer
  5uL   10x Taq Ligase Buffer
  8uL   50mM MgCl2
  4uL   2.5mM dNTPs
      +
  .5uL  ExoIII diluted to 10 U/uL
  2uL   Phusion HF Polymerase (courtesy of Finnzyme)@ 2 U/uL
  4uL   Taq Ligase @ 40 U/uL
      +
  Water to 50uL


We kept this reaction mix at 50C for 60 seconds to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C at a very slow ramp rate the now-single-stranded parts anneal to one another, allowing the Phusion polymerase and Taq ligase to fill in and closed the gaps. Because the yields are too low to clone directly from this reaction we PCRed full constructs using BB prefix and suffix primers and high fidelity polymerase which could then be cloned into a biobrick-compatible plasmid.