Composite

Part:BBa_K1132038

Designed by: iGEM Toulouse   Group: iGEM13_INSA_Toulouse   (2013-09-20)

XOR-inverted RFP gate (BBa_K1132035) with T7 polymerase under the control of a strong promoter

DO NOT USE this part! The FimE sites did not work well, this design can be use by replace K137008 by K1077001 and K137010 by K1077000.

This BioBrick is design to test the XOR gate (BBa_K1132004) by measuring the level of RFP after recombination events. The biobrick BBa_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites. The T7 polymerase gene is also present in the biobrick, under the control of a strong promoter, strong RBS. This part can therefore be used stand-alone as all elements to control the RFP output are present.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1116
    Illegal NheI site found at 1139
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 178
    Illegal AgeI site found at 290
  • 1000
    COMPATIBLE WITH RFC[1000]


In vitro characterization

The basic idea is to perform the switch with a cell lysis containing the recombinase and the plasmid containing the gate to be switched. After incubation, transformation of the plasmid containing the gate allows the quantification of switched versus non switched plasmids. Experiments have been performed as described in the protocol. [[1]]

Results

In the absence of recombinase or in recombination assays with Bxb1, the resulting colonies are white. Therefore, non-specific recombinases cannot switch the XOR gate. When XOR was put in the presence of FimE, the presence of red colonies validated the switch. However, only 5% of the plasmids were switched. Hence, the switch of XOR using FimE was not highly efficient. Several explanations can be made: i) low expression of FimE; ii) low activity of FimE in the tested buffer; iii) low activity of FimE on our designed gate. Improvement on FimE protocols or redesign of new sites could have been performed with our Michigan buddies but, unfortunately, time was running out.

X2.PNG

We also performed a second switch, using the XOR gate that has been switched by FimE. We prepared the DNA from the XOR gate that has been previously switched by FimE (a red colony). This plasmid DNA was tested in the presence of PhiC31 recombinase. The picture below demonstrates that the second switch occurred: white colonies are now present.

XOR2_result.PNG

Xor_2_swiche_par_Fim_E_avec_PhiC31.PNG


We also tried the switch with PhiC31 only. We used the original Siuti’s plasmid containing PhiC31, but no switch was obtained. The results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.

This part can be considered as a good amelioration of the RFP reporter system. Reporter systems can be controlled either by regular positive or negative regulators. Such regulation systems obey to biological rules and are modulated between to limit values. The logic gates obey a second type of rule, closer to the electronic description of the 0 and 1 (Off and On) states. Therefore, the switch are less prone to physiological states than regulatable promoters. Furthermore, we also demonstrated that the switch are permanent and can be genetically transmitted to the offspring. This also constitutes a new system for determining at any time any occurring event that can further disappear. The switch would serve as a tracer. Last, as these switches obey Boolean logic operations, detection of independent, time separated events can also be achieved. A full description of the logic gates is present on our Wiki.

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