Team:ETH Zurich/Decoder/Results

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Contents

PABA generator

Cloning

Fig.1: Plasmid map of <partinfo>BBa_K909014</partinfo> in pSB1C3.

We were succesful in constructing both parts <partinfo>BBa_K909014</partinfo> (2699bp) and <partinfo>BBa_K909015</partinfo> (2656bp) based on the already existing parts <partinfo>BBa_K137055</partinfo> and <partinfo>BBa_S04039</partinfo>. The vector backbone is <partinfo>pSB1C3</partinfo> (2079bp). They differ from each other due to the fact that <partinfo>BBa_K909014</partinfo> contains the constitutive promoter <partinfo>BBa_J23100</partinfo> with two NheI restriction sites.

To verify the sizes of the constructs, the plasmids containing <partinfo>BBa_K909014</partinfo> and <partinfo>BBa_K909015</partinfo> were digested with

1. NheI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2685 bp & 2061 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 4724 bp

2. XbaI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2721 bp & 2048 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 2678 bp & 2048 bp
Fig.2:Plasmid map of <partinfo>BBa_K909015</partinfo> in pSB1C3.


Fig.3.: Digests left to right: Ladder, <partinfo>BBa_K909014</partinfo> (NheI/PstI), <partinfo>BBa_K909014</partinfo> (XbaI,PstI),<partinfo>BBa_K909015</partinfo> (NheI/PstI), <partinfo>BBa_K909015</partinfo> (XbaI,PstI).








As it is visible in Fig.3. the band sizes of the <partinfo>BBa_K909014</partinfo> & <partinfo>BBa_K909015</partinfo> digestion match the expectation. In a further step <partinfo>BBa_K909015</partinfo> is joined to the PL hybrid promoter <partinfo>BBa_K909011</partinfo> for implementation.


Hybrid promoters

= Assembly part 1

In a first step the following two promoters were cloned together:


After assembling all parts for the decoder, we want to test the circuit by simulating blue and red light with aTc and IPTG. Our output system is based on fluorescent proteins (eYFP, mCherry and eCFP) giving us the possibility to analyse the result by single cell analysis (FACS).

Fig.3.: Digests left to right: Ladder, 3 screened colonies containing the decoder plasmid.


Fig.3.: Digests left to right: Ladder, 3 screened colonies containing the decoder plasmid.
Pseva 183.jpg


Fig.4: Boolean logic for testing of Decoder.


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