Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization

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Latest revision as of 20:01, 5 October 2011

The system is customizable!!

Customization of the Plug 'n' Play with DNA assembly standard opens the door to a wide range of applications.

When do you need to customize the system?

When a desired biological part is not yet a part of the Plug'n'Play with DNA kit.

When seamless assembly is required (e.g. protein fusion).

When the introduction of mutations are required.

When the introduction of deletions are required.

When short sequences such as linkers, RBSs, signal peptides etc. are required.

A guide for the most common design requests is given below. The guide is based on work conducted by Morten H. H. Nørholm and Hansen et. al. Figures obtained from Genee et al. (in preparation).

Creating new parts

Creating new biological parts can be necessary, when they are not already included in the Plug 'n' Play kit and submitted to the Registry of Standard Biological Parts.

1. To create a new part, forward and reverse primers have to be designed for the DNA of interest. The free-ware netprimer is an excellent tool for finding suitable primers.

2. In accordance with the Plug’n’Play assembly system the category of the part i.e. promoter, GOI, TS, module, or marker cassette has to be determined.

3. Subsequently the linkers belonging to the category have to be identified. The appropriate linkers and Plug'n'Play tail for the part can be found in the linker table. The forward and reverse primer linkers are 8-9 bp long, and are placed at the 5’-end of the primers.

5. Order the primers from your favourite oligo company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.

6. After receiving the primers, you are ready to perform the PCR (see protocol Amplification of biobricks by PCR ). Be aware that the addition of the linker to the primer may increase the melting temperature of the primer.

7. Check the band size of the PCR product on an agarose-gel. The new BioBrick can be used directly or purified by agarose-gel purification.

8. The assembly of the new BioBricks should be performed according to the protocol USER cloning.

9. You should now have assembled one new plasmid or linear device.

Seamless assembly

Seamless assembly is often required when constructing fusion protein or if large genetic parts have to be assembled in pieces, e.g. when constructing complex enzymes and synthases for fungi.

1. If you want to design a construct where all parts are seamlessly assembled go directly to step 2. Otherwise the standard linkers should be added to the parts that do not need to be fused seamless according to the linker table. For more details see the 'creating new parts' section.

2. To design the primers for the seamless assembly the software PHUSER can be used. This software was created by the DTU iGEM team from 2009.

3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.

4. To amplify the BioBricks the protocol Amplification of biobricks by PCR can be used.

5. Check the PCR reaction on an agarose-gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification.

6. The assembly of new BioBricks is executed according to the protocol USER cloning.

7. A scar free BioBrick device or plasmid should now have been assembled.

Point mutations

The introduction of point mutations can have different purposes, for instance the removal of undesired restriction site or for alteration of a proteins catalytic function. When introducing mutations it is only necessary for the primers to overlap in the complementary region (linker). Depending on whether the point mutation should be degenerate or not only one or both primers needs to carry the mutation. Degeneration means that more than one codon may code for the same amino acid. Thereby, a degenerate mutation can give different outcomes in use of tRNA's.

Introducing a point mutation with non-degenerate codons
Both primers have to carry the desired mutation to ensure that the codon and the resulting amino acid are generated by the mutation.

1. To introduce a point mutation in a vector, the mutation has to be defined first.

2. To design the primers the software PHUSER can be used. Remember to introduce the desired mutation into the primer. Two primers are used per mutation.

3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.

4. To amplify the BioBricks the protocol Amplification of biobricks by PCR can be used.

5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.

6. The assembly of new BioBricks should be performed according to the protocol USER cloning.

7. A BioBrick, device or plasmid with the desired point mutation should now have been assembled.

Introducing a degenerate point mutation, which can give a different codon usage
Only one primer has to carry the desired point mutation. The procedure of assembly is the same as for the non-degenerate point mutation above.

It is also possible to introduce several point mutations in different regions by customizing the Plug 'n' Play assembly standard. This only requires one round of PCR and one round of cloning. The difference is that the PCR fragments made for each mutation will have to be fused together meaning that the introduction of two mutations would require the fusion of three PCR fragments, and so on.

Deletions

With this guide a gene or part of a gene or any other desired sequence of a plasmid, device, or BioBrick can be deleted.

1. First the site of the deletion should be defined.

2. The primers need to be designed so that the fusion point will bridge two desired sections of the gene at the matching USER linkers.

3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.

4. To amplify the BioBricks the protocol Amplification of biobricks by PCR can be used.

5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.

6. The assembly of new BioBricks should be performed according to the protocol USER cloning.

7. A BioBrick, device or new plasmid with the deletion should now have been assembled.

Adding short sequences

Short sequences such as ribosomal binding sites, kozak sequences etc. can be added by incorporating them into the primer that will be used to amplify the part it should be fused with. Introducing a short sequence can also be of interest when two proteins are wished to be connected by a linker. The linker can also carry a single point mutation, be degenerate or non-degenerate, which means that only one or both primers needs to carry the mutation. When creating a linker a short sequence of either random or known sequence is added by incorporating the sequence into the forward or reverse primers.

Introducing a linker

1. When introducing a linker in the connection with a gene of interest (GOI), the first step is to define the mutation.

2. Primers are designed for the defined mutation site. To introduce the desired mutation one nucleotide should be changed in the primer. Four primers are used per mutation.

3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.

4. To amplify the BioBricks the protocol Amplification of biobricks by PCR can be used.

5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed.

6. The assembly of new BioBricks and parts should be performed according to the protocol USER cloning.

The figure below shows the process of creating a degenerate linker (illustrated with a loop). Primers can be designed by using the standard Plug’n’Play linkers.

Illustrated below the process of creating a non-degenerate linker.

Example

Synthetic biology is all about creativity, and sometimes the standard is just not enough, making it necessary to combine the above mentioned customizations. The figure below illustrates that "Plug 'n' Play with DNA" makes it possible to assemble a degenerate linker (red), a non-degenerate linker (yellow), introduce a non-degenerate point mutation (X) as well as a degenerate point mutation (N) into one plasmid in just ONE round of cloning.

Linker table

Tips & Tricks

Design primers så the Tms between forward and reverse primer are within 2-5°C.

Remember that the annealing temperature should normally be 3°C under the Tm. However, when the linker is added to the primer, the Tm is increased. We have experienced that the best results are obtain when the final annealing temperature is above 62°C, if the primer have a Tm around 59°C to begin with.

The X7 Phusion polymerase have in the PCR reaction a extension time of 1000bp per minut.

References

[1] Nørholm, M.H.H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21, 2010.

[2] Hansen, B. G.; Salomonsen, B.; Nielsen, M. T.; Nielsen, J. B.; Hansen, N. B.; Nielsen, K.F.; Regueira, T. B.; Nielsen, J.; Patil, K. R.; Mortensen, U. H.; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.

[3] Genee HJ, Bonde MT, Bagger FO, Olsen LR. PHUSER v2 (In preparation).

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 <div class="text">
 <font color=" #990000" face="arial" size="6">
-<br>
+<br><br>
 <b>The system is customizable!!</b><br> <br>
 </font>
-</div>
-We know how important flexibility within molecular biology is, however setting up a standard often entails rigidity. Therefore we here provide a guide on how to customize the Plug’n’Play assembly standard. All procedures only require one round of PCR and one round of USER cloning ensuring a fast construction time. The customization of the system is based on the papers by Morten Nørholm and Hansen et. al. and the work performed by the iGEM team DTU Denmark from 2009.
-<br>
 <table cellpadding=5px>
-<th>
+<tr>
-<td width="25%" height="100%" valign="top" valign="center" >
+<td height="100%" valign="top" valign="center" >
 <br>
 <div id="menu">
 <!--<span id="test">test</span>-->
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#creating new biobricks " class="h1">Creating new parts</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#When do you need to customize the system?" class="h1"> When do you need to customize the system?</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Seamless assembly " class="h1">Seamless assembly</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Seamless assembly" class="h1"> Seamless assembly</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Point mutations " class="h1">Point mutations</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Point mutations" class="h1"> Point mutations</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Deletions " class="h1">Deletions</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Deletions" class="h1"> Deletions</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Adding short sequences " class="h1">Adding short sequences</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Adding short sequences" class="h1"> Adding short sequences</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Example " class="h1">Example</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Example" class="h1"> Example</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Linker table " class="h1">Linker table</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Linker table" class="h1"> Linker table</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Tips & Tricks " class="h1">Tips & Tricks</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Tips & Tricks" class="h1"> Tips & Tricks</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#References " class="h1">References</a><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#References" class="h1"> References</a><br>
 </div>
 </td>
+<td width="75%" height="100%" valign="top">
 <br>
-<b>When do you need to customize the system?</b><br>
-</p>
-  </font>
 <p align="justify">
+Customization of the Plug 'n' Play with DNA assembly standard opens the door to a wide range of applications.</p>
-•	When a desired biological part is not yet a part of the Plug’n’Play with DNA kit
-<br>
-•	When seamless assembly is required (e.g. protein fusion)
 <br>
-•	When you want to introduce mutations
+<a name="When do you need to customize the system?"></a><h2><b>When do you need to customize the system?</b></h2>
-<br>
-•	When you want to introduce deletions
-<br>
-•	When you want to add short sequences such as an RBS, signal peptide etc.
+<p align="justify">
+<dt>
+<dd><li>When a desired biological part is not yet a part of the Plug'n'Play with DNA kit. </li></dd>
+<dd><li>When seamless assembly is required (e.g. protein fusion).</li></dd>
+<dd><li>When the introduction of mutations are required.</li></dd>
+<dd><li>When the introduction of deletions are required.</li></dd>
+<dd><li>When short sequences such as linkers, RBSs, signal peptides etc. are required.</li></dd>
+</dt>
+<br><br>
+A guide for the most common design requests is given below. The guide is based on work conducted by Morten H. H. Nørholm and Hansen et. al. Figures obtained from Genee et al. (in preparation).
+</p>
-<td width="40%" height="100%" valign="top">
-<br>
 <br>
 <a name="Creating new parts"></a><h2><b>Creating new parts</b></h2>
+<p align="justify">
+Creating new biological parts can be necessary, when they are not already included in the Plug 'n' Play kit and submitted to the Registry of Standard Biological Parts.</p><br>
+<p align="justify">
+<dt>
+<dd>1.	To create a new part, forward and reverse primers have to be designed for the DNA of interest. The free-ware <a href="http://www.premierbiosoft.com/netprimer/index.html">netprimer</a> is an excellent tool for finding suitable primers.</dd>
+<dd>2.	In accordance with the Plug’n’Play assembly system the category of the part i.e. promoter, GOI, TS, module, or marker cassette has to be determined.</dd>
+<dd>3.	Subsequently the linkers belonging to the category have to be identified. The appropriate linkers and Plug'n'Play tail for the part can be found in the <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Linker table">linker table</a>. The forward and reverse primer linkers are 8-9 bp long, and are placed at the 5’-end of the primers. </dd>
+<dd>5.	Order the primers from your favourite oligo company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.</dd>
+<dd>6.	After receiving the primers, you are ready to perform the PCR (see protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#PCR">Amplification of biobricks by PCR</a> ). Be aware that the addition of the linker to the primer may increase the melting temperature of the primer. </dd>
+<dd>7.	Check the band size of the PCR product on an agarose-gel. The new BioBrick can be used directly or purified by agarose-gel purification. </dd>
+<dd>8.	The assembly of the new BioBricks should be performed according to the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER cloning"> USER cloning</a>. </dd>
+<dd>9.	You should now have assembled one new plasmid or linear device.</dd>
+</dt>
+</p>
 <br>
-.	To create a new part you first have to design primers as you normally do it for the DNA of interest. The free-ware netprimer can assist you in finding suitable primers.<br><br>
-.	To make the part compatible with the Plug’n’Play assembly standard you have to determine the category of the part i.e. promoter, GOI, TS, module, or marker cassette.<br><br>
-.	Once you have done this you have to identify the category in the linker table to find the appropriate Plug’n’Play tails for your part. In the last column of the table, the tails that you should put in front of your forward and reverse primers can be found.<br><br>
-.	You simply take these 8-9 base sequences at place them at the 5’-end of your primers. <br><br>
-.	Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
-.	When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
-.	After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarosegelpurification. <br><br>
-.	Now you are ready to assemble your favorite parts by following the protocol USER cloning. <br><br>
-.	Congratulations you should now have your desired construct!
-<br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
-<br>
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
-<img src="https://static.igem.org/mediawiki/2011/f/f8/Koncept_igem_forside_1.png" height="200px" > </img> &nbsp;
+</a>
-<br>
-<br>
-<br><br>
 <a name="Seamless assembly"></a><h2><b>Seamless assembly</b></h2>
+<p align="justify">
+Seamless assembly is often required when constructing fusion protein or if large genetic parts have to be assembled in pieces, e.g. when constructing  complex enzymes and synthases for fungi.</p>
+<br>
+<p align="justify">
+<dt>
+<dd>1.	If you want to design a construct where all parts are seamlessly assembled go directly to step 2. Otherwise the standard linkers should be added to the parts that do not need to be fused seamless according to the <a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/PlugnplayAssembly/customization#Linker table">linker table</a>. For more details see the 'creating new parts' section.</dd>
+<dd>2.	To design the primers for the seamless assembly the software <a href="http://www.cbs.dtu.dk/services/PHUSER/">PHUSER</a> can be used. This software was created by the DTU iGEM team from 2009.</dd>
+<dd>3.	The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.</dd>
+<dd>4.	To amplify the BioBricks the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#PCR">Amplification of biobricks by PCR</a> can be used. </dd>
+<dd>5.	Check the PCR reaction on an agarose-gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification. </dd>
+<dd>6.	The assembly of new BioBricks is executed according to the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER cloning"> USER cloning</a>. </dd>
+<dd>7.	A scar free BioBrick device or plasmid should now have been assembled.</dd>
+</p>
 <br>
-.	Decide how many parts you will have to assemble scar-free. Depending on if this part constitutes a single part, several parts, or an entire device select the corresponding linkers from the linker table and put these linkers on the forward and reverse primers corresponding to your final construct.<br><br>
+<p align="center">
-.	To design the primers for the scar-free assembly the software PHUSER developed by the DTU iGEM team from 2009 can be used.<br><br>
-.	Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
-.	When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
-.	After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarosegelpurification. <br><br>
-.	Now you are ready to assemble your favorite parts by following the protocol USER cloning. <br><br>
-.	Congratulations you should now have joined your biobricks without leaving a scar construct!<br><br>
-<img src="https://static.igem.org/mediawiki/2011/6/6d/Customization_seemless_assembly.png" height="200px" > </img> &nbsp;
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
+</a>
+</p>
 <br>
 <br>
@@ Line 136: / Line 195: @@
 <a name="Point mutations"></a><h2><b>Point mutations</b></h2>
+<p align="justify">
+The introduction of point mutations can have different purposes, for instance the removal of undesired restriction site or for alteration of a proteins catalytic function. When introducing mutations it is only necessary for the primers to overlap in the complementary region (linker). Depending on whether the point mutation should be degenerate or not only one or both primers needs to carry the mutation. Degeneration means that more than one codon may code for the same amino acid. Thereby, a degenerate mutation can give different outcomes in use of tRNA's.</p><br>
+<p align="justify">
+<b>Introducing a point mutation with non-degenerate codons</b><br>
+Both primers have to carry the desired mutation to ensure that the codon and the resulting amino acid are generated by the mutation.</p><br>
+<p align="justify">
+<dt>
+<dd>1. To introduce a point mutation in a vector, the mutation has to be defined first.</dd>
+<dd>2. To design the primers the software <a href="http://www.cbs.dtu.dk/services/PHUSER/">PHUSER</a> can be used. Remember to introduce the desired mutation into the primer. Two primers are used per mutation.</dd>
+<dd>3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.</dd>
+<dd>4. To amplify the BioBricks the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#PCR">Amplification of biobricks by PCR</a> can be used. </dd>
+<dd>5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed. </dd>
+<dd>6.	The assembly of new BioBricks should be performed according to the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER cloning">USER cloning</a>. </dd>
+<dd>7.	A BioBrick, device or plasmid with the desired point mutation should now have been assembled.</dd>
+</dt>
+</p>
 <br>
-When introducing the mutation it is only neccessary for the primers to overlap in the complementary region (linker) and only one primer needs to carry the mutation.
+<p align="center">
-<br><br>
-<b>Introducing point mutations in vectors</b><br><br>
-. Define the mutation you want to introduce in your vector<br><br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
+</a>
+</p>
-. Design primers to anneal at the site where you want to introduce the mutation by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutation simply by changing a nucleotide to the one you wish. For one mutation you in total need two primers.<br><br>
+<br>
+<p align="justify">
+<b>Introducing a degenerate point mutation, which can give a different codon usage</b><br>
+Only one primer has to carry the desired point mutation. The procedure of assembly is the same as for the non-degenerate point mutation above.</p><br>
+<p align="center">
-. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
-. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
+</a>
+</p>
-. After checking the PCR reaction on an agarosegel you can use your new biobrick directly or if needed you can purify the PCR product by agarose gel purification. <br><br>
+<br>
+<p align="justify">
+It is also possible to introduce several point mutations in different regions by customizing the Plug 'n' Play assembly standard. This only requires one round of PCR and one round of cloning. The difference is that the PCR fragments made for each mutation will have to be fused together meaning that the introduction of two mutations would require the fusion of three PCR fragments, and so on.</p><br>
-. Now you are ready to assemble your vector by following the protocol USER cloning. <br><br>
-. Congratulations you should now have introduced a point mutation into your vector.<br><br>
-<img src="https://static.igem.org/mediawiki/2011/b/b9/Customization_point-mutation_not_degenerate.png" with="200px"height="200px alt="""/>
+<a name="Deletions"></a><h2><b>Deletions</b></h2>
+<p align="justify">
+With this guide a gene or part of a gene or any other desired sequence of a plasmid, device, or BioBrick can be deleted. <br><br>
+<dt>
+<dd>1. First the site of the deletion should be defined.</dd>
+<dd>2. The primers need to be designed so that the fusion point will bridge two desired sections of the gene at the matching USER linkers.</dd>
+<dd>3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.</dd>
+<dd>4. To amplify the BioBricks the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#PCR">Amplification of biobricks by PCR</a> can be used. </dd>
+<dd>5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed. </dd>
+<dd>6.	The assembly of new BioBricks should be performed according to the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER cloning">USER cloning</a>. </dd>
+<dd>7. A BioBrick, device or new plasmid with the deletion should now have been assembled.</p></dd>
+</dt>
-<br><br><br>
-<b>Introducing point mutations in a gene of interest</b><br><br>
+<p align="center">
-. Define the mutation you want to introduce in the gene of interest (GOI)<br><br>
-. Design primers to anneal at the site where you want to introduce the mutation by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutation simply by changing a nucleotide to the one you wish. For one mutation you in total need four primers.<br><br>
-. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
-. When you receive your primers you set up the two PCR reactions, we recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
+</a>
-. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification. <br><br>
+</p>
-. Now you are ready to assemble your biobricks by following the protocol USER cloning. <br><br>
-. Congratulations you should now have introduced a point mutation into your GOI.<br><br>
+<br><br>
-<img src="https://static.igem.org/mediawiki/2011/c/cb/Customization_linker_not_degenerate.png" with="200px"height="200px alt="""/>
+<a name="Adding short sequences"></a><h2><b>Adding short sequences</b></h2>
+<p align="justify">
+Short sequences such as ribosomal binding sites, kozak sequences etc. can be added by incorporating them into the primer that will be used to amplify the part it should be fused with.
+Introducing a short sequence can also be of interest when two proteins are wished to be connected by a linker. The linker can also carry a single point mutation, be degenerate or non-degenerate, which means that only one or both primers needs to carry the mutation.
+When creating a linker a short sequence of either random or known sequence is added by incorporating the sequence into the forward or reverse primers.</p><br>
-<br><br><br>
+<b>Introducing a linker</b><br>
+<p align="justify">
+<dt>
+<dd>1. When introducing a linker in the connection with a gene of interest (GOI), the first step is to define the mutation.</dt>
+<dd>2. Primers are designed for the defined mutation site. To introduce the desired mutation one nucleotide should be changed in the primer. Four primers are used per mutation.</dt>
+<dd>3. The primers are ordered from your favourite company. Notice that the price can be a bit higher and delivery time a bit longer, when ordering primers with a uracil incorporated. We have had a good experience with the price, delivery time and quality of the primers ordered from Integrated DNA Technology.</dt>
+<dd>4. To amplify the BioBricks the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#PCR">Amplification of biobricks by PCR</a> can be used. </dt>
+<dd>5. Check the PCR reaction on an agarose gel before assembly. The new BioBricks can be used directly or purified by agarose-gel purification if needed. </dt>
+<dd>6.	The assembly of new BioBricks and parts should be performed according to the protocol <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER cloning">USER cloning</a>. </dt>
+</dt>
+<p align="justify">
+The figure below shows the process of creating a degenerate linker (illustrated with a loop). Primers can be designed by using the standard Plug’n’Play linkers.</p> <br>
+<p align="center">
-<b>Introducing several point mutations</b><br><br>
-It is possible to introduce several point mutations in different regions of interest by customizing the Plug’n’Play assembly standard. This still only requires one round of PCR and one round of cloning, the only difference is that more PCR fragments will have to be fused. This means that the introduction of two mutations would require the fusion of three PCR fragments, and so on.<br><br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
+</a>
-. Define the mutations you want to introduce.<br><br>
-. Design primers to anneal at the sites where you want to introduce mutations by using the software PHUSER developed by the DTU iGEM team from 2009. In the primers you should introduce the desired mutations simply by changing the nucleotides to the ones you wish. <br><br>
+</p>
+<br><br>
+Illustrated below the process of creating a non-degenerate linker.<br><br>
+<p align="center">
-. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
+</a>
-. When you receive your primers you set up the PCR reactions. We recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
+</p>
-. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification. <br><br>
+<br>
-. Now you are ready to assemble your biobricks by following the protocol USER cloning. <br><br>
+<a name="Example"></a><h2><b>Example</b></h2>
+<p align="justify">
+Synthetic biology is all about creativity, and sometimes the standard is just not enough, making it necessary to combine the above mentioned customizations. The figure below illustrates that "Plug 'n' Play with DNA" makes it possible to assemble a degenerate linker (red), a non-degenerate linker (yellow), introduce a non-degenerate point mutation (X) as well as a degenerate point mutation (N) into one plasmid in just ONE round of cloning. </p> <br>
-. Congratulations you should now have introduced several point mutations. <br><br>
+<p align="center">
+<a href="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" rel="lightbox" title="">
+  <img src="https://static.igem.org/mediawiki/igem.org/7/79/Mathilde.png" alt="" height="200px" />
+</a>
-<img src="https://static.igem.org/mediawiki/2011/f/fc/Customization_point-mutation_degenerate.png" with="200px"height="200px alt="""/>
+</p>
 <br><br>
-<a name="Deletions"></a><h2><b>Deletions</b></h2>
+<a name="Linker table"></a><h2><b>Linker table</b></h2>
 <br>
-. Define where you want to introduce a deletion.<br><br>
-. Design primers to anneal at the sites where you want to introduce the deletion by using the software PHUSER developed by the DTU iGEM team from 2009. The primers should be designed in such a way that on the fusion point will bridge two desired sections of the gene at the matching USER linkers.<br><br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/a/ac/Linkertable.png" with="400px"height="400px"></img>
+</p>
+<br>
+<br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/f/fe/Overveiw_tails_2.png" height="100px"> </img>
+</p>
-. Next you order the primers from your favorite company. You should notice that ordering primers with a uracil incorporated increases the price of your primers, Integrated DNA Technology offers a fair price for such primers.<br><br>
+<br>
+<br>
-. When you receive your primers you set up the PCR reactions. We recommend you to use our protocol Amplification of biobricks by PCR. <br><br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/2/27/Overveiw_module_tails-TS.png" height="100px" > </img>
+</p>
-. After checking the PCR reactions on an agarosegel you can use your new biobricks directly or if needed you can purify the PCR products by agarose gel purification. <br><br>
-. Now you are ready to assemble your biobricks by following the protocol USER cloning. <br><br>
-. Congratulations you should now have introduced a deletion. <br><br>
-<img src="https://static.igem.org/mediawiki/2011/e/ee/Customization_deletion.png" with="200px"height="200px alt="""/>
+<br><br>
+<a name="Tips & Tricks"></a><h2><b>Tips & Tricks</b></h2>
+<dd>
+<li> Design primers så the Tms between forward and reverse primer are within 2-5°C.</li>
+<li> Remember that the annealing temperature should normally be 3°C under the Tm. However, when the linker is added to the primer, the Tm is increased. We have experienced that the best results are obtain when the final annealing temperature is above 62°C, if the primer have a Tm around 59°C to begin with.</li>
+<li> The X7 Phusion polymerase have in the PCR reaction a extension time of 1000bp per minut.</li>
+</dd>
 <br><br>
-<a name="Adding short sequences"></a><h2><b>Adding short sequences</b></h2>
+<a name="References"></a><h2><b>References</b></h2>
-<br>
-A short sequence is simply added by incorporating the sequence into the forward or reverse primer. This has been illustrated with a loop in the figure below. Primers can be designed by using the standard Plug’n’Play linkers or by using the software PHUSER. <br><br>
+[1] Nørholm, M.H.H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21, 2010.<br><br>
-<img src="https://static.igem.org/mediawiki/2011/a/a3/Customization_linker_degenerate.png" with="200px"height="200px alt="""/>
+[2] Hansen, B. G.; Salomonsen, B.; Nielsen, M. T.; Nielsen, J. B.; Hansen, N. B.; Nielsen, K.F.; Regueira, T. B.; Nielsen, J.; Patil, K. R.; Mortensen, U. H.; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.<br><br>
+[3] Genee HJ, Bonde MT, Bagger FO, Olsen LR. PHUSER v2 (In preparation).
 <br><br>
-<a name="Example"></a><h2><b>Example</b></h2>
-<br>
-Synthetic biology is all about creativity, here we show an example of what can be done when using the standard is just not enough for you! The figure below shows that you in one cloning round can assemble a degenerate linker with a non-degenerate linker (yellow), introduce a point mutation non-degenerate with a point mutation degenerate.
-<img src="https://static.igem.org/mediawiki/2011/9/9d/Customization_combination.png" with="200px"height="200px alt="""/>
-<br><br>
+</ul>
+</td>
+</tr>
+</table>
+</div>
-<a name="Linker table"></a><h2><b>Linker table</b></h2>
+<div id="EndOfPage"></div>
-<br>
-<center><img src="https://static.igem.org/mediawiki/2011/a/ac/Linkertable.png" with="400px"height="400px alt="""/></center>
-<br><br>
-<a name="Tips & Tricks"></a><h2><b>Tips & Tricks</b></h2>
-<br>
-Try to design your primers such that their Tm's are within 2 degrees celicus of each other
-<br><br><br>
-<a name="References"></a><h2><b>References</b></h2>
-<br>
-Nørholm, M. H. H. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol. 10, 21 (2010).<br>
-Hansen, Bjarke G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.<br><br>
-https://2009.igem.org/Team:DTU_Denmark
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