Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system/library and selection"

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We decided to create a tRNA/aaRS library in two different ways, showing the advantages and disadvantages of both methods.
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The first method is based on primers, which contain a randomization in a certain position. Both forward and reverse primer own this randomization. When the library plasmid is amplified with these primers it results in plasmids containing a variety of different amino acids in this certain position. While the execution of this method is easy, the diversity of plasmid sequences is not large. That is because of the statistical loss of different versions due to the required binding of two complementary primers on the same DNA fragment.
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/0/0b/T--Bielefeld-CeBiTec--NNK_PCR.png">
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<p class="figure subtitle"><b>Figure 2: Creation of a library</b><br> Creation of a library using a randomized primers and PCR (Polymerase Chaine Reaction).</p>
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Revision as of 07:58, 11 October 2017

Library and Selection

Generating the library

The integration of a new amino acid in the translational cycle implies the creation of a large number of tRNA/aminoacyl tRNA synthetase (tRNA/aaRS) variants. This can be achieved by generating a library. This library serves as a basis for selective processes or screenings, where an optimal tRNA/aaRS can be obtained. The library is based on the pSB1C3 plasmid, with the tyrosyl-tRNA synthetase (TyrRS) under control of a glnS promoter inserted between the iGEM BioBrick prefix and suffix. For the cloning of the vector, we used the primers 17jj and 17jk for the amplification of the pSB1C3 backbone and the primers 17hq and 17ht for the amplification of the TyrRS.

Figure 1: Library plasmid pSB1C3 containing the tyrosyl tRNA synthetase.


We decided to create a tRNA/aaRS library in two different ways, showing the advantages and disadvantages of both methods. The first method is based on primers, which contain a randomization in a certain position. Both forward and reverse primer own this randomization. When the library plasmid is amplified with these primers it results in plasmids containing a variety of different amino acids in this certain position. While the execution of this method is easy, the diversity of plasmid sequences is not large. That is because of the statistical loss of different versions due to the required binding of two complementary primers on the same DNA fragment.

Figure 2: Creation of a library
Creation of a library using a randomized primers and PCR (Polymerase Chaine Reaction).