Team:Alberta/Optimization
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<p>Much work has been done to try and increase the efficiency by which we generate the Bytes, anchor them, assemble them, and terminate them. A general outline of the optimizations we have considered and worked on are shown below as well as their effects on the process. | <p>Much work has been done to try and increase the efficiency by which we generate the Bytes, anchor them, assemble them, and terminate them. A general outline of the optimizations we have considered and worked on are shown below as well as their effects on the process. | ||
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<h4>The dNTPs</h4> | <h4>The dNTPs</h4> | ||
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- | <li><b>Concentration effects of dNTPs</b> were considered. We ran six PCR reactions in total: 3 sets for each AB/BA primer pair. The three conditions tested were 0.2, 0.6, 0.8, 1.2, and 1.5 | + | <li><b>Concentration effects of dNTPs</b> were considered. We ran six PCR reactions in total: 3 sets for each AB/BA primer pair. The three conditions tested were 0.2, 0.6, 0.8, 1.2, and 1.5 µM final concentration of dNTPs. Optimal PCR yield was at 0.2 µM dNTPs. |
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- | An essential step in assembly with BioBytes | + | An essential step in assembly with BioBytes is the preparation of the Bytes. Following PCR the product is USER<sup>TM</sup> digested to nick the DNA. Finally, the Bytes are purified away from these small ssDNA pieces to prevent their binding to the sticky ends during assembly and consequently negatively influencing the efficiency of construction. The following describes the results of optimization experiments conducted to increase efficiency of BioBytes. |
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<h4>USER<sup>TM</sup> Digestion</h4> | <h4>USER<sup>TM</sup> Digestion</h4> | ||
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- | <li>The <b>amount of USER<sup>TM</sup></b> in the digest was investigated by digesting two sets of BA/AB Bytes: either 4% or 10% total volume of USER<sup>TM</sup> was added to the PCR product tubes. The 4%, for instance, corresponds to 1 | + | <li>The <b>amount of USER<sup>TM</sup></b> in the digest was investigated by digesting two sets of BA/AB Bytes: either 4% or 10% total volume of USER<sup>TM</sup> was added to the PCR product tubes. The 4%, for instance, corresponds to 1 µL of USER<sup>TM</sup> mix in a 25 µL PCR reaction. The conclusion was that slight improvement in ligation efficiency was seen for 10% over 4% USER<sup>TM</sup> addition. |
<li><b>USER<sup>TM</sup> digestion duration</b> was considered at 0.5, 1, 1.5, and 2 hours. By examining the efficiency of ligation of AB and BA Bytes processed under these conditions that no changes in efficiency were observed after 1 hour. | <li><b>USER<sup>TM</sup> digestion duration</b> was considered at 0.5, 1, 1.5, and 2 hours. By examining the efficiency of ligation of AB and BA Bytes processed under these conditions that no changes in efficiency were observed after 1 hour. | ||
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- | <li>The effect of <b>BioByte Excesses</b> on the efficiency of assembly was investigated by binding 5 | + | <li>The effect of <b>BioByte Excesses</b> on the efficiency of assembly was investigated by binding 5 µmol of anchor version#2 (see the <a href="https://2009.igem.org/Team:Alberta/DNAanchor">Anchor/Terminator</a> section upon which 1x, 2x, and 5x excesses of a second Byte were added. There was a clear and drastic improvement in ligation efficiency and thus as large a molar excess of successive Bytes should be added to the growing construct as one can muster. |
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- | <li>The <b>volume of washes</b> between Byte addition to the beads is of concern. Large washes tend to slow bead pelleting and thus leads to loss of beads since some will be aspirated off. On the other hand, small washes are not effective. By simple experimentation we found that 75 | + | <li>The <b>volume of washes</b> between Byte addition to the beads is of concern. Large washes tend to slow bead pelleting and thus leads to loss of beads since some will be aspirated off. On the other hand, small washes are not effective. By simple experimentation we found that 75 µL washes of 40 µL worth of 4 mg mL<sup>-1</sup> beads works the best. |
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- | <li>The <b>number of washes</b> was optimized by binding Bytes to anchor bound beads. The aspirated washes were saved and run on a gel. Seven consecutive 75 | + | <li>The <b>number of washes</b> was optimized by binding Bytes to anchor bound beads. The aspirated washes were saved and run on a gel. Seven consecutive 75 µL washes with wash/binding buffer were done. It was evident that after even just one wash most of the excess DNA was gone and just two washes removes all traces of excess DNA. |
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<h4>uPASTE</h4> | <h4>uPASTE</h4> | ||
- | <p>Due to the need for the obscure DNA polymerase Pfu Cx and the USER<sup>TM</sup> mix as well as the money and time to use these things, it was an impetus to try to develop an alternative to the current BioBytes method. The alternative that was being developed was dubbed uPASTE (<b> | + | <p>Due to the need for the obscure DNA polymerase Pfu Cx and the USER<sup>TM</sup> mix as well as the money and time to use these things, it was an impetus to try to develop an alternative to the current BioBytes method. The alternative that was being developed was dubbed uPASTE (<b>U</b>racil <b>P</b>CR <b>A</b>mplified <b>St</b>icky <b>E</b>nds). It was hoped that the sticky ends could be generated in the PCR step, foregoing the rest of the processing steps of the current system. Pfu contains a uracil binding domain that prevents it's reading through of uracil containing DNA. We figured we could use this to terminate PCR with primers containing a single deoxyuracil. However, further research and consultation showed us why the system failed after even significant attempts to optimize the PCR. Pfu polymerase binds uracils irreversibly, thus inhibiting the polymerase. </p> |
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Latest revision as of 00:45, 21 November 2009
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Optimizing Linear AssemblyMuch work has been done to try and increase the efficiency by which we generate the Bytes, anchor them, assemble them, and terminate them. A general outline of the optimizations we have considered and worked on are shown below as well as their effects on the process. |
The Uracil Dilemma
The original format of the universal primers did not have the uracils distributed evenly within the primer. The result was poor efficiency in construction on a bead. Our hypothesis was that the uracils, if they were distributed more evenly, would create smaller pieces of ssDNA that would more easily melt off the Byte to generate fully ssDNA 12 base overhangs. The first version of our USER ends is shown below. By changing primers to their current form we have consequently increased efficiency of construction 2.5 times that of the first version.
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PCR OptimizationThe first step in producing workable quantities of BioBytes is PCR with the universal deoxyuracil-containing primers. Only slight tweaking of PCR conditions was required to produce ideal quantities of Bytes.
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BioByte ProcessingAn essential step in assembly with BioBytes is the preparation of the Bytes. Following PCR the product is USERTM digested to nick the DNA. Finally, the Bytes are purified away from these small ssDNA pieces to prevent their binding to the sticky ends during assembly and consequently negatively influencing the efficiency of construction. The following describes the results of optimization experiments conducted to increase efficiency of BioBytes.
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BioByte AssemblyOnce the Bytes have been amplified, digested, and purified they can be assembled onto the bead. The process of building the constructs on the bead is simple but critical since by this point you have invested money and time into getting your Bytes ready. Thus it was important to optimize the assembly as much as we could in the short duration of the project.
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Alternatives to the USERTM SystemOriginally, project BioBytes required the use of a few different restriction enzymes to generate the 12 base 3' sticky ends. Also, we had experimented with termination PCR to generate our ends using the universal deoxyuracil primers. However, the current system of BioBytes remains the most effective to date. Alternatives to the current system are still being considered.
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