Difference between revisions of "Part:BBa K1493702"

Latest revision as of 15:15, 23 October 2014

Toggle switch part with reporter gene gfp

Part of the [http://2014.igem.org/Team:Wageningen_UR/project/kill-switch#toggleswitchplasmid BananaGuard toggle switch] containing a gfp reporter gene. Combined with the BioBrick BBa_K1493703 this part forms a toggle switch. This BioBrick is tested as the whole toggle switch. The part is assembled with BBa_K1493703 in a pSB3K3 backbone.

Usage and Biology

To determine if the toggle switch is functional and the repressor proteins produced under the promoters inhibit the gene expression of each other the system was characterized. Three cultures were prepared in M9 medium in duplo, inoculated with Escherichia coli strain NEB5α carrying the toggle switch with reporter. The first containing 500 ng/ml aTc to get the cells into the active state and the second inducing the resting state by 2 mM IPTG as described by Gardner et al[1]. A third culture is grown without inducer, evoking a random state. E. coli carrying pSB3K3 with CI and lacI with no promoter was used as an auto fluorescence control. Cultures were grown overnight and fluorescence was measured the next morning using a plate reader (395 excitation, 509 emission). Cells did not grow well in M9 with 2mM IPTG, as the OD was low (OD 600 nm of 0.3) compared to OD 600 nm 0.6-0.7 of the cultures grown with aTc or with no inducer, after overnight incubation.

‘’Figure 1. The relative fluorescence unit of each toggle switch state. Fluorescence is measured in duplo of cell cultures carrying the pSB3K3 plasmid with the toggle switch construct (BBa_K1493702, BBa_K1493703) grown in M9 medium containing 500 ng/ml aTc (green), 2 mM IPTG (red) and with no inducer added to the medium (blue).’’

Figure 1 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1227
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1903

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1493702 short</partinfo>
-Part of the Banana Guard toggle switch containing a reporter gene. For the orther part of the toggle switch that combines with this part see BBa_K1493703.
+Part of the [http://2014.igem.org/Team:Wageningen_UR/project/kill-switch#toggleswitchplasmid BananaGuard toggle switch] containing a ''gfp'' reporter gene. Combined with the BioBrick <partinfo>BBa_K1493703</partinfo> this part forms a toggle switch.
+This BioBrick is tested as the whole toggle switch. The part is assembled with <partinfo>BBa_K1493703</partinfo> in a <partinfo>pSB3K3</partinfo> backbone.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+To determine if the toggle switch is functional and the repressor proteins produced under the promoters inhibit the gene expression of each other the system was characterized. Three cultures were prepared in M9 medium in duplo, inoculated with ''Escherichia coli'' strain NEB5α carrying the toggle switch with reporter. The first containing 500 ng/ml aTc to get the cells into the active state and the second inducing the resting state by 2 mM IPTG as described by Gardner et al[1]. A third culture is grown without inducer, evoking a random state. ''E. coli'' carrying <partinfo>pSB3K3</partinfo> with CI and lacI with no promoter was used as an auto fluorescence control. Cultures were grown overnight and fluorescence was measured the next morning using a plate reader (395 excitation, 509 emission). Cells did not grow well in M9 with 2mM IPTG, as the OD was low (OD 600 nm of 0.3) compared to OD 600 nm 0.6-0.7 of the cultures grown with aTc or with no inducer, after overnight incubation.
+https://static.igem.org/mediawiki/2014/5/5a/Characterization_Toggle_Switch.png
+‘’Figure 1. The relative fluorescence unit of each toggle switch state. Fluorescence is measured in duplo of cell cultures carrying the <partinfo>pSB3K3</partinfo> plasmid with the toggle switch construct (<partinfo>BBa_K1493702</partinfo>, <partinfo>BBa_K1493703</partinfo>) grown in M9 medium containing 500 ng/ml aTc (green), 2 mM IPTG (red) and with no inducer added to the medium (blue).’’
+Figure 1 shows that the cultures grown in M9 with 500 ng/ml aTc have an average relative fluorescence unit of 7300. The cultures grown with 2 mM IPTG give an average fluorescence unit of 1000. The cells grown in M9 with no inducer give a fluorescence of 5200 RFU. These values indicate that the toggle switch is functional as it has a high fluorescence when grown with aTc, which means it is in its active state. The resting state is reached with low fluorescence when grown with IPTG. We can conclude from the RFU value of the cultures grown with no inducer that the culture has a mix of both states.
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