Difference between revisions of "Part:BBa K3128017:Experience"

 
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===Applications of BBa_K3128017===
 
===Applications of BBa_K3128017===
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=='''Materials and Methods'''==
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===Bacterial Strain===
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<div style="text-align:justify;">
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The assays are made with streptomycin resistant '''BTH101''' <i>E.Coli</i> strain, which are <i>cya-</i> bacteria.<br>
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In this strain, the endogenous adenylate cyclase gene has been deleted in order to obtain a bacterium that is <u>unable to produce endogenous '''cAMP'''</u>,
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thus avoiding the presence of potential false positives and making the system more sensitive.
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</div>
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===Design of the plasmids===
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<div style="text-align:justify;"> 
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For the mBACTH, as three biobricks have to be inserted in the bacterium to constitute the entire system, genetic constructions have been made in order to co-transform only two compatible plasmids:
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<i>pOT18-Nlc</i> contains '''OmpX gene fused to the T18''' sub-part and the '''NanoLuciferase gene''' under the control of the '''plac promoter'''; it has an ampicillin resistant gene and the pMB1 replication origin.<br>
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<i>pOT18-Nlc</i> contains [https://parts.igem.org/Part:BBa_K3128001 NanoLuciferase reporter for BACTH assay] and [https://parts.igem.org/Part:BBa_K3128017 OmpX WT protein fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter]</u>.<br>
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<i>pOT25</i> contains '''OmpX gene fused to the T25 subpart'''. It has a kanamycin resistant gene and the p15A replication origin.<br>
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<i>pOT25</i> contains [https://parts.igem.org/Part:BBa_K3128018 OmpX WT protein fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter].</u><br>
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'''Those constructs will be the negative condition that show the background noise of the initial mBACTH system.'''<br>
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<br>
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<i>pOT18-Nlc-ZIP</i> is similar to <i>pOT18-Nlc</i> with the addition of a '''leucine-zipper''' sequence between the '''OmpX signal peptide''' and the '''OmpX gene'''.<br>
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<i>pOT18-Nlc-ZIP</i> contains [https://parts.igem.org/Part:BBa_K3128001 NanoLuciferase reporter for BACTH assay] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128026 OmpX WT protein fused with LZ and T18 subpart of Bordetella Pertussis AC under constitutive promoter].<br>
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<i>pOT25-ZIP</i> is similar to <i>pOT25</i> with the addition of a '''leucine-zipper''' sequence between the '''OmpX signal peptide''' and the '''OmpX gene'''.
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<i>pOT25-ZIP</i> contains [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128027 OmpX WT protein fused with LZ and T25 subpart of Bordetella Pertussis AC under constitutive promoter].<br>
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'''Those constructs will be the positive condition that show how the signal increases if both sub-parts are brought together with the mBACTH.'''<br>
  
 
===User Reviews===
 
===User Reviews===

Revision as of 15:31, 7 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3128017

Materials and Methods

Bacterial Strain

The assays are made with streptomycin resistant BTH101 E.Coli strain, which are cya- bacteria.
In this strain, the endogenous adenylate cyclase gene has been deleted in order to obtain a bacterium that is unable to produce endogenous cAMP, thus avoiding the presence of potential false positives and making the system more sensitive.

Design of the plasmids

For the mBACTH, as three biobricks have to be inserted in the bacterium to constitute the entire system, genetic constructions have been made in order to co-transform only two compatible plasmids:

pOT18-Nlc contains OmpX gene fused to the T18 sub-part and the NanoLuciferase gene under the control of the plac promoter; it has an ampicillin resistant gene and the pMB1 replication origin.
pOT18-Nlc contains NanoLuciferase reporter for BACTH assay and OmpX WT protein fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter</u>.
pOT25 contains OmpX gene fused to the T25 subpart. It has a kanamycin resistant gene and the p15A replication origin.
pOT25 contains OmpX WT protein fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter.</u>
Those constructs will be the negative condition that show the background noise of the initial mBACTH system.


pOT18-Nlc-ZIP is similar to pOT18-Nlc with the addition of a leucine-zipper sequence between the OmpX signal peptide and the OmpX gene.
pOT18-Nlc-ZIP contains NanoLuciferase reporter for BACTH assay and OmpX WT protein fused with LZ and T18 subpart of Bordetella Pertussis AC under constitutive promoter.
pOT25-ZIP is similar to pOT25 with the addition of a leucine-zipper sequence between the OmpX signal peptide and the OmpX gene. pOT25-ZIP contains OmpX WT protein fused with LZ and T25 subpart of Bordetella Pertussis AC under constitutive promoter.
Those constructs will be the positive condition that show how the signal increases if both sub-parts are brought together with the mBACTH.

User Reviews

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UNIQf789f225d378de85-partinfo-00000001-QINU