Part:BBa_K1132031
Test of the AND gate (BBa_K1132001) with RFP
This BioBrick is designed to test the AND gate (BBa_K1132001) 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 assembled with our gate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 286
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 295
Illegal AgeI site found at 1091
Illegal AgeI site found at 1203 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 315
Illegal BsaI.rc site found at 476
In vitro characterization of BBa_K1132031
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. http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb
Results
In the absence of recombinase, the colonies are white. As a result, AND gate does not have transcriptional leakage and non-specific recombinases cannot switch the door.
We tested the AND gate with Bxb1. After incubation with Bxb1, the colonies are still white (this is normal!), therefore visual inspection of the switch was not possible. The only possibility was to sequence independent clones We analyzed five independent colonies by sequencing, all of them were switched.
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 [http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/logic_gates Wiki].
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