Team:Alberta/DNAanchor

From 2009.igem.org

(Difference between revisions)
Line 46: Line 46:
<h3>Anchoring System</h3>
<h3>Anchoring System</h3>
-
<p>The current method for Byte production (ie: USER<sup>TM</sup>) necessitated a particular 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. Once the desired number of Bytes 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 1</B>.</P>
 
-
<p>A vital component of the BioBytes method is the use of a biotinylated DNA anchor in order to allow unidirectional assembly of the Bytes on paramagnetic beads by sequestering the 5' ends of Bytes, leaving only the 3' ends available to bind incomding Bytes.  The anchor itself has three vital components:  A 5’ biotinylation, a double stranded DNA (dsDNA) portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the anchor.  Our team has considered a number anchor systems, each with their own set of advantages and disadvantages.  The three main anchor systems we investigated are summarized below. See <B>Table 1</B>.</P>
+
<p>A vital component of the BioBytes method is the use of a biotinylated DNA Anchor in order to allow unidirectional assembly of the Bytes on paramagnetic beads by sequestering the 5' ends of Bytes, leaving only the 3' ends available to bind incomding Bytes.  The Anchor itself has three vital components:  A 5’ biotinylation, a double stranded DNA (dsDNA) portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the Anchor.  Our team has considered a number anchor systems, each with their own set of advantages and disadvantages.  The three main anchor systems we investigated are summarized below. See <B>Table 1</B>.</P>
<br>
<br>
Line 57: Line 56:
<br>
<br>
-
<p>The current BioBytes anchor system utilizes a 5’-biotin which anchors the construct to beads by binding non-covalently, but with great strength, to the covalently linked streptavidin on the surface of the paramagnetic beads.  There is also a 5’-15 nucleotide spacer region of ssDNA which facilitates more efficient binding of the anchor to the bead as the binding pocket of streptavidin is deep and thus a highly flexible ssDNA linker is recommended to allow the biotin to effectively bind into this deep pocket.  A 21 bp double stranded portion of the anchor contains the I-SceI recognition sequence, which when digested with I-SceI produces 4 base overhangs, but also includes four deoxyuracil residues.  These uracils are excised by New England Biolab’s USER<sup>TM</sup> system to generate single nucleotide gaps in the top strand.  USER<sup>TM</sup> digestion thus effectively destroys the anchor and produces an 18 base 3’ overhang which becomes important for recircularization of the construct.  Finally the anchor contains the A or B 3’ overhangs complementary to those of the Bytes, allowing their binding to the anchor.
+
<p>The current BioBytes anchor system utilizes a 5’-biotin which anchors the construct to beads by binding non-covalently, but with great strength, to the covalently linked streptavidin on the surface of the paramagnetic beads.  There is also a 5’-15 nucleotide spacer region of ssDNA which facilitates more efficient binding of the Anchor to the bead as the binding pocket of streptavidin is deep and thus a highly flexible ssDNA linker is recommended to allow the biotin to effectively bind into this deep pocket.  A 21 bp double stranded portion of the Anchor contains the I-SceI recognition sequence, which when digested with I-SceI produces 4 base overhangs, but also includes four deoxyuracil residues.  These uracils are excised by New England Biolab’s USER<sup>TM</sup> system to generate single nucleotide gaps in the top strand.  USER<sup>TM</sup> digestion thus effectively destroys the Anchor and produces an 18 base 3’ overhang which becomes important for recircularization of the construct.  Finally the Anchor contains the A or B 3’ overhangs complementary to those of the Bytes, allowing their binding to the Anchor. See <B>Figure 1</B>.</P>
-
. See <B>Figure 1</B>.</P>
+
<br>
<br>
Line 67: Line 65:
<br>
<br>
 +
<h3>Termination System</h3>
 +
 +
<p>Once the construct has been completed, i.e. the last Byte has been added, the construct may be released from the beads as is by a simple I-SceI digestion or a USER<sup>TM</sup> digestion, thus yielding a linear construct.  If a circular construct, such as a plasmid, is desired then a final "Terminator" piece must be added.  This piece is similar in construction to the Anchor, whereby there is a dsDNA I-SceI recognition sequence with four deoxyuracils incorporated into it on one strand, as well as an A or B 3' overhang.  The Terminator binds to the last Byte and release is once again achieved by I-SceI digestion or USER<sup>TM</sup> digestion.  In either case both the Anchor and Terminator develop sticky ends that are complementary to eachother: 4 bases if I-SceI digestion is utilized, or 18 bases if USER is used.  USER<sup>TM</sup> digestion is obviously preferred since 18 bp of interaction will form spontaneously and without ligation, and thus transformation of the construct can proceed immediately. See <B>Figure 1</B>.</P>
       </div></div>
       </div></div>

Revision as of 06:35, 21 October 2009

University of Alberta - BioBytes










































































































DNA Anchor/Terminator

Anchoring System

A vital component of the BioBytes method is the use of a biotinylated DNA Anchor in order to allow unidirectional assembly of the Bytes on paramagnetic beads by sequestering the 5' ends of Bytes, leaving only the 3' ends available to bind incomding Bytes. The Anchor itself has three vital components: A 5’ biotinylation, a double stranded DNA (dsDNA) portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the Anchor. Our team has considered a number anchor systems, each with their own set of advantages and disadvantages. The three main anchor systems we investigated are summarized below. See Table 1.


Table 1: Overview of three different anchoring systems that were considered for BioBytes. 5' Biotin (BTN), single stranded DNA (ssDNA), nucleotide (nt), double stranded DNA (dsDNA).


The current BioBytes anchor system utilizes a 5’-biotin which anchors the construct to beads by binding non-covalently, but with great strength, to the covalently linked streptavidin on the surface of the paramagnetic beads. There is also a 5’-15 nucleotide spacer region of ssDNA which facilitates more efficient binding of the Anchor to the bead as the binding pocket of streptavidin is deep and thus a highly flexible ssDNA linker is recommended to allow the biotin to effectively bind into this deep pocket. A 21 bp double stranded portion of the Anchor contains the I-SceI recognition sequence, which when digested with I-SceI produces 4 base overhangs, but also includes four deoxyuracil residues. These uracils are excised by New England Biolab’s USERTM system to generate single nucleotide gaps in the top strand. USERTM digestion thus effectively destroys the Anchor and produces an 18 base 3’ overhang which becomes important for recircularization of the construct. Finally the Anchor contains the A or B 3’ overhangs complementary to those of the Bytes, allowing their binding to the Anchor. See Figure 1.


Figure 1: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions.


Termination System

Once the construct has been completed, i.e. the last Byte has been added, the construct may be released from the beads as is by a simple I-SceI digestion or a USERTM digestion, thus yielding a linear construct. If a circular construct, such as a plasmid, is desired then a final "Terminator" piece must be added. This piece is similar in construction to the Anchor, whereby there is a dsDNA I-SceI recognition sequence with four deoxyuracils incorporated into it on one strand, as well as an A or B 3' overhang. The Terminator binds to the last Byte and release is once again achieved by I-SceI digestion or USERTM digestion. In either case both the Anchor and Terminator develop sticky ends that are complementary to eachother: 4 bases if I-SceI digestion is utilized, or 18 bases if USER is used. USERTM digestion is obviously preferred since 18 bp of interaction will form spontaneously and without ligation, and thus transformation of the construct can proceed immediately. See Figure 1.

Retrieved from "http://2009.igem.org/Team:Alberta/DNAanchor"