Difference between revisions of "Part:BBa K1132037"

Latest revision as of 10:09, 14 November 2013

AND-inverted RFP gate (BBa_K1132034) 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 AND gate (BBa_K1132034) 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 1141
Illegal NheI site found at 1164
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 957
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 264
Illegal AgeI site found at 376
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. 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, non-specific recombinases cannot switch the AND gate.

We tested the AND gate with FimE. After incubation with FimE, 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 due to lack of time we did not have the results. We analyzed five independent colonies by sequencing, but none was switched. Several hypotheses can be made. First, the presence of the PhiC31 switching sequences may inhibit.

The AND was also tested with the Siuti’s construction parts containing PhiC31, but not switched has been obtained. 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 [http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/logic_gates Wiki].

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1132037 short</partinfo>
+'''''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 AND gate ([https://parts.igem.org/Part:BBa_K1132034 BBa_K1132034]) by measuring the level of RFP after recombination events. The biobrick [https://parts.igem.org/Part:BBa_K081014 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.