Difference between revisions of "Team:UIUC Illinois/Project"
| Line 120: | Line 120: | ||
</b></h> | </b></h> | ||
| − | <li>Primer Design | + | <li>1.<i>Primer Design</i> <br> |
| − | + | When designing the primers, ensure that adjacent segments in the plasmid have identical sequences on the ends. The insert sequence and the vector sequence should be compatible with each other. These identical sequences can be created via PCR with primers containing a 5’ end identical to the adjacent segment and a 3’ end that would anneal to the target sequence. Once the primers have been designed, amplify the amount of primer DNA by PCR. </li> | |
| − | <li>Check the purity and concentration of the PCR amplification and/or restriction digest. If the product is found to be impure, perform a gel purification to remove impurities. </li> | + | <p>Traditional cloning relied on sticky ends created by restriction sites, which normally provide 4 bp overhangs. Gibson Assembly improves on this aspect by providing a larger overhang via the primers designed to amplify the DNA vector and DNA insert. This technique generates a 20-30 bp overhang. |
| + | </p> | ||
| + | <li>2.Check the purity and concentration of the PCR amplification and/or restriction digest. If the product is found to be impure, perform a gel purification to remove impurities. </li> | ||
</ol> | </ol> | ||
<br> | <br> | ||
<ol> | <ol> | ||
| − | <h style="text-align:center;"><b> | + | <h style="text-align:center;"><b>Preparation before performing Gibson Assembly: </b></h> |
<p> | <p> | ||
| − | For our method, we used NEB’s Gibson Assembly Master Mix. The Master Mix consists of three enzymes in a single buffer: | + | For our method, we used NEB’s Gibson Assembly Master Mix. The Master Mix consists of three enzymes in a single buffer with the following functions: |
| − | <ul>1. T5 exonuclease | + | <ul>1. T5 5’ exonuclease, which chews back the 5’ end of the DNA to create a 3’ overhang so that the complementary strands could anneal to each other.</ul> |
| − | <ul>2. Phusion DNA Polymerase | + | <ul>2.Phusion DNA Polymerase, which incorporates nucleotides to fill in the gaps in the annealed DNA fragment</ul> |
| − | <ul>3. Taq DNA ligase | + | <ul>3. Taq DNA ligase, which anneals the DNA fragments and removes the ‘nicks’ and ‘scars’. |
| + | |||
| + | </ul> | ||
</p> | </p> | ||
| Line 137: | Line 141: | ||
<br> | <br> | ||
| − | <p>The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. | + | <p>The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. The appropriate amount of DNA, when combining 2-3 fragments in a Gibson Assembly reaction, is 0.02 – 0.5 pmol of total DNA. Cloning efficiency is best when 50 -100 ng of vector is used, with 2-3 equivalents of insert in a 20 µl reaction. Sample should then be incubated in a thermocycler at 50ºC for 15 minutes. After 15 minutes, the assembly should be complete. </p> |
| − | The appropriate amount of DNA, when combining 2-3 fragments in a Gibson Assembly reaction, is 0.02 – 0.5 pmol of total DNA. Cloning efficiency is best when 50 -100 ng of vector is used, with 2-3 equivalents of insert in a 20 µl reaction. Sample should then be incubated in a thermocycler at 50ºC for 15 minutes, | + | |
| Line 167: | Line 170: | ||
<br id="description"> | <br id="description"> | ||
<h1 id="homeH">Experiment Description</h1> | <h1 id="homeH">Experiment Description</h1> | ||
| − | <p> One of the biggest obstacles to scientific advancement and discovery is the | + | <p>One of the biggest obstacles to scientific advancement and discovery is the exorbitantly high price for conducting simple experiments. To put this in perspective, cloning 2-3 fragments of DNA using Gibson assembly costs $18.50. Currently, the purchase of one Gibson Assembly Kit for ten reactions is $185.00. For research that heavily depends on assembling many DNA fragments, the bill can rise very quickly.</p><br> |
| − | <p> The aim of creating a homemade Gibson Assembly recipe was to drastically lower the prices of performing Gibson Assembly. The price could be lowered if the same outcome could be performed using unpurified enzymes. The unpurified enzymes would replace the purified enzymes commonly used in the Gibson Assembly master mix. | + | <p>The aim of creating a homemade Gibson Assembly recipe was to drastically lower the prices of performing Gibson Assembly. The price could be lowered if the same outcome could be performed using unpurified enzymes. The unpurified enzymes would replace the purified enzymes commonly used in the Gibson Assembly master mix. </p><br> |
| − | + | <p>The idea of our project can be summarized in one sentence “using Gibson assembly to make enzymes for Gibson assembly.” </p><br> | |
| − | + | <p>To expand on this statement, the Gibson assembly cloning method was used to create constructs that contained the genes for Pyrococcus furiosus DNA polymerase and Thermotoga maritima DNA ligase. These constructs were created so that when they are transformed into DH5α cells, the local cell machinery would be used for high expression of the genes and produce an abundant number of T. maritima DNA ligase and P. furiosus DNA polymerase enzymes.</p><br> | |
| − | <p>The idea of our project can be summarized in one sentence “using Gibson assembly to make enzymes for Gibson | + | |
| − | <p>To expand on this statement, the Gibson assembly cloning method was used to create constructs that contained the genes for DNA polymerase and DNA ligase. These constructs were created so that when they are transformed into DH5α cells, the local cell machinery would be used for high expression of the | + | <p>Once colonies of the transformed cells grow, cell lysate will be made in buffer similar to the Gibson assembly buffer. Two tubes of cell lysate, one containing T. maritima DNA ligase and the other containing P. furiosus DNA polymerase, will be added in ratios relative to each other to the reaction tubes. The ratio that yields results will be determined empirically. The background T5 5’ exonuclease naturally present in the lysate would account for the T5 5’ exonuclease needed for the reaction. |
| − | <p>Once colonies of the transformed cells grow, cell lysate will be made in buffer similar to the Gibson assembly buffer. | + | |
| − | <p>To verify that the isothermal reaction using unpurified enzyme worked, | + | </p><br> |
| − | + | <p>To verify that the isothermal reaction using unpurified enzyme worked, the allegedly assembled construct must be transformed into DH5α cells. The DNA can then be purified from the bacterial colonies and sent to the UIUC Core Sequencing facility for sequencing. If the returned sequence matches the known sequence of the insert, we can conclude that the isothermal reaction was successful.</p><br> | |
| − | + | ||
| + | |||
</div> | </div> | ||
Revision as of 23:38, 1 November 2017
Background
Gibson Assembly is a revolutionary method for assembling multiple linear DNA fragments (original paper) by Dr. Daniel Gibson at the J. Craig Venter Institute in 2009. Multiple overlapping DNA fragments can be joined by a single reaction regardless of the fragment length, which adds to the versatility of the method. By adding the three different enzymes (T5 5’ exonuclease, Phusion DNA polymerase, and Taq DNA ligase), a fully ligated double-stranded DNA molecule is assembled. This method is proven to be efficient due to the ease of the reaction – needing only one tube of reaction – and the effectiveness of the reaction: no scars on the ligated DNA, non-selective compatibility of DNA fragments, and no specific restriction sites are needed.
- 1.Primer Design
When designing the primers, ensure that adjacent segments in the plasmid have identical sequences on the ends. The insert sequence and the vector sequence should be compatible with each other. These identical sequences can be created via PCR with primers containing a 5’ end identical to the adjacent segment and a 3’ end that would anneal to the target sequence. Once the primers have been designed, amplify the amount of primer DNA by PCR. - 2.Check the purity and concentration of the PCR amplification and/or restriction digest. If the product is found to be impure, perform a gel purification to remove impurities.
Traditional cloning relied on sticky ends created by restriction sites, which normally provide 4 bp overhangs. Gibson Assembly improves on this aspect by providing a larger overhang via the primers designed to amplify the DNA vector and DNA insert. This technique generates a 20-30 bp overhang.
For our method, we used NEB’s Gibson Assembly Master Mix. The Master Mix consists of three enzymes in a single buffer with the following functions:
- 1. T5 5’ exonuclease, which chews back the 5’ end of the DNA to create a 3’ overhang so that the complementary strands could anneal to each other.
- 2.Phusion DNA Polymerase, which incorporates nucleotides to fill in the gaps in the annealed DNA fragment
- 3. Taq DNA ligase, which anneals the DNA fragments and removes the ‘nicks’ and ‘scars’.
The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. The appropriate amount of DNA, when combining 2-3 fragments in a Gibson Assembly reaction, is 0.02 – 0.5 pmol of total DNA. Cloning efficiency is best when 50 -100 ng of vector is used, with 2-3 equivalents of insert in a 20 µl reaction. Sample should then be incubated in a thermocycler at 50ºC for 15 minutes. After 15 minutes, the assembly should be complete.
Experiment Description
One of the biggest obstacles to scientific advancement and discovery is the exorbitantly high price for conducting simple experiments. To put this in perspective, cloning 2-3 fragments of DNA using Gibson assembly costs $18.50. Currently, the purchase of one Gibson Assembly Kit for ten reactions is $185.00. For research that heavily depends on assembling many DNA fragments, the bill can rise very quickly.
The aim of creating a homemade Gibson Assembly recipe was to drastically lower the prices of performing Gibson Assembly. The price could be lowered if the same outcome could be performed using unpurified enzymes. The unpurified enzymes would replace the purified enzymes commonly used in the Gibson Assembly master mix.
The idea of our project can be summarized in one sentence “using Gibson assembly to make enzymes for Gibson assembly.”
To expand on this statement, the Gibson assembly cloning method was used to create constructs that contained the genes for Pyrococcus furiosus DNA polymerase and Thermotoga maritima DNA ligase. These constructs were created so that when they are transformed into DH5α cells, the local cell machinery would be used for high expression of the genes and produce an abundant number of T. maritima DNA ligase and P. furiosus DNA polymerase enzymes.
Once colonies of the transformed cells grow, cell lysate will be made in buffer similar to the Gibson assembly buffer. Two tubes of cell lysate, one containing T. maritima DNA ligase and the other containing P. furiosus DNA polymerase, will be added in ratios relative to each other to the reaction tubes. The ratio that yields results will be determined empirically. The background T5 5’ exonuclease naturally present in the lysate would account for the T5 5’ exonuclease needed for the reaction.
To verify that the isothermal reaction using unpurified enzyme worked, the allegedly assembled construct must be transformed into DH5α cells. The DNA can then be purified from the bacterial colonies and sent to the UIUC Core Sequencing facility for sequencing. If the returned sequence matches the known sequence of the insert, we can conclude that the isothermal reaction was successful.
