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

From 2011.igem.org

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We know how important flexibility within molecular biology is, however setting up a standard often entails rigidity.  
We know how important flexibility within molecular biology is, however setting up a standard often entails rigidity.  
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<br>We therefore here provide a guide on how to customize the Plug’n’Play assembly standard.
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<br>We therefore here provide a guide on how to customize the Plug’n’Play assembly standard. All procedures require 1 round of PCR and 1 round of assembly.
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<br>
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• When you want to introduce mutations
• When you want to introduce mutations
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<br>
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• When you want to add short sequences such as an RBS, signal peptide etc.
<td width="75%" height="100%" valign="top">
<td width="75%" height="100%" valign="top">
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<a name="Creating new parts"></a><h2><b>Creating new parts</b></h2>
<a name="Creating new parts"></a><h2><b>Creating new parts</b></h2>
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<br>
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Creating new parts:<br><br>
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1. 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>
 +
2. 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>
 +
3. Once you have done this you have to identify the category in table 1 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>
 +
4. You simply take these 8-9 base sequences at place them at the 5’-end of your primers. <br><br>
 +
5. 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>
 +
6. 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>
 +
7. 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>
 +
8. Now you are ready to assemble your favorite parts by following the protocol USER cloning. <br><br>
 +
9. Congratulations you should now have your desired construct!
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<a name="Seamless assembly"></a><h2><b>Seamless assembly</b></h2>
<a name="Seamless assembly"></a><h2><b>Seamless assembly</b></h2>
<br>
<br>
 +
Seamless assembly:<br><br>
 +
 +
1. 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 table 1 and put these linkers on the forward and reverse primers corresponding to your final construct.<br><br>
 +
2. 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>
 +
3. 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>
 +
4. 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>
 +
5. 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>
 +
6. Now you are ready to assemble your favorite parts by following the protocol USER cloning. <br><br>
 +
7. 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;
<img src="https://static.igem.org/mediawiki/2011/6/6d/Customization_seemless_assembly.png" height="200px" > </img> &nbsp;

Revision as of 07:45, 16 September 2011



The system is customizable!!
We know how important flexibility within molecular biology is, however setting up a standard often entails rigidity.
We therefore here provide a guide on how to customize the Plug’n’Play assembly standard. All procedures require 1 round of PCR and 1 round of assembly.


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 scar-free assembly is required (e.g. protein fusion)
• When you want to introduce mutations
• When you want to add short sequences such as an RBS, signal peptide etc.




Creating new parts


Creating new parts:

1. 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.

2. 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.

3. Once you have done this you have to identify the category in table 1 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.

4. You simply take these 8-9 base sequences at place them at the 5’-end of your primers.

5. 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.

6. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR.

7. 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.

8. Now you are ready to assemble your favorite parts by following the protocol USER cloning.

9. Congratulations you should now have your desired construct!

 



Seamless assembly


Seamless assembly:

1. 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 table 1 and put these linkers on the forward and reverse primers corresponding to your final construct.

2. To design the primers for the scar-free assembly the software PHUSER developed by the DTU iGEM team from 2009 can be used.

3. 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.

4. When you receive your primers you set up a PCR reaction, we recommend you to use our protocol Amplification of biobricks by PCR.

5. 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.

6. Now you are ready to assemble your favorite parts by following the protocol USER cloning.

7. Congratulations you should now have joined your biobricks without leaving a scar construct!

 

Site-directed mutagenesis












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