Difference between revisions of "Part:BBa K3128017"
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=Team Grenoble-Alpes 2019=
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<partinfo>BBa_K3128017 short</partinfo>
  
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=='''Materials and Methods'''==
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==Sequence and features==
 +
<div style="text-align:justify;"> 
 +
This biobrick is composed of the external membrane [https://parts.igem.org/Part:BBa_K3128031 Outer Membrane Protein X Wild Type <i>(OmpX-WT)</i>]
 +
protein fused at its C-terminal end to a 54 aa [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128010 Gly-Gly-Ser Linker (GGS)]
 +
followed by the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1638004 T18 sub-part of the <i>Bordetella Pertussis</i> adenylate cyclase] (AC) .<br>
 +
This biobrick is under a constitutive promoter [https://parts.igem.org/Part:BBa_J23101 J23101],
 +
thereby OmpX-WT fused to T18 protein is constitutively expressed leading to a large amount of recombinant protein in the outer membrane at any time.<br>
 +
 
 +
<br>This BioBrick is intended to operate with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128018 BBa_K3128018].<br>
 +
These two biobricks are the negative condition of the '''mBACTH''' <i>-free sub-parts condition-</i>.<br>
 +
OmpX proteins are fused to the adenylate cyclase sub-parts at their C-terminal ends. The fusion protein '''move freely''' in the bacterial outer membrane,
 +
but they are not forced to get closer by any mean. <br>
 +
The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.<br>
 +
The signal measured is considered as background noise.
  
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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.
 
 
</div>
 
</div>
  
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===Design of the plasmids===
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<div style="text-align:justify;">
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<partinfo>BBa_K3128017 SequenceAndFeatures</partinfo>
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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:
+
  
 
<br>
 
<br>
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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].<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>
 
−
'''Those constructs will be the negative condition that show the background noise of the initial mBACTH system.'''<br>
 
  
 +
==Usage and Biology==
 +
<div style="text-align:justify;"> 
 +
In 1989, Fields and Song demonstrated a new genetic system allowing the detection of protein-protein interaction <sup>(1)</sup>. At first, it was performed in ''Saccharomyces cerevisiae''
 +
yeast and it was named the yeast two-hybrid assay (Y2H). In 1998, Ladant and al. described the system in bacteria <sup>(2)</sup>. Nowadays, this biological technique is mostly
 +
used to show and characterize the physical interaction between two cytosolic proteins or internal membrane proteins <i>in vivo</i> <sup>(3)</sup>.
 
<br>
 
<br>
−
<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'''. <br>
 
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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>
 
  
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https://2019.igem.org/wiki/images/thumb/f/f9/T--Grenoble-Alpes--mBACTH_plamides.png/800px-T--Grenoble-Alpes--mBACTH_plamides.png
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<br>
 +
===<u>Bacterial Adenylate Cyclase Two-Hybrid</u> (BACTH)===
 +
<div style="text-align:justify;"> 
  
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===Transformation===
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<br>The principle lies on the interaction-mediated reconstitution of a signalling cascade in <i>Escherichia coli</i>. The messenger molecule involved in this cascade is the
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For the assay with the membrane BACTH, BTH101 are co-transformed either with <u><i>pOT18-Nlc</i> and <i>pOT25</i> plasmids</u> : '''negative condition''',<br>  
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cyclic adenosine monophosphate (cAMP) produced by the adenylate cyclase. Adenylate cyclase is an enzyme catalysing the cAMP production from ATP. It
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or <u><i>pOT18-Nlc-ZIP</i> and <i>pOT25-ZIP</i> plasmids</u> : '''positive condition'''.<br>
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physiologically participates to the cellular transmission. <br>
 +
 
 +
<br>This system involves the <i>Bordetella pertussis</i> '''adenylate cyclase''' which is the responsible agent for the pertussis disease.
 +
Adenylate cyclase catalytic domain has the particularity to be splittable in two distinct parts: '''T18''' and '''T25''' sub-parts, <u>unable to fonction unless they
 +
reassociate.</u> Each sub-part of the enzyme is fused with a protein of interest, either the bait or the prey protein chose beforehand by the experimentator. <br>
 +
 
 +
<span style="margin: 12%;">
 +
https://2019.igem.org/wiki/images/e/ec/T--Grenoble-Alpes--BACTH_classicBACTH.gif
 +
</span>
 +
 
 +
<br>If two proteins interact, then '''T18''' and '''T25''' are bring together and reconstitute a <u>functional adenylate cyclase enzyme</u> thus enabling cAMP production. Using
 +
<i>cya-</i> bacteria<i> – strain for whom the adenylate gene is deleted, involving an absence of this endogenous enzyme – </i> a BACTH could be done with the creation of two
 +
fusion proteins : the first one, fused at its N or C terminal intracellular end with the '''T18 sub-part'''; the second one fused with the '''T25 sub-part'''. <br>
 +
The interaction of these proteins of interest will lead to the adenylate cyclase reconstitution, thus <u>initiating cAMP production</u>. The cAMP produced will act as a
 +
messenger by fixing itself to the transcriptional activator CAP, cAMP form the <u>CAP-cAMP</u> complex, controlling the expression of the lactose promoter by '''initiating transcription of the following gene'''. <br>
 +
This promoter is placed upstream the chosen reporter gene.<br>
 +
</div>
  
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===The assay===
 
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<div style="text-align:justify;">
 
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To be able to test the membrane BACTH (mBACTH), '''OmpX''' proteins have been muted to be able to integrate an unnatural amino acid in one of their extracellular loops by implementing the amber stop codon TAG.
 
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A specific tRNA can then add this azide-functionalized amino acid in the protein, which is able to fix a DIBO group - these modified proteins are called '''COMPs'''. <br>
 
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COMPs are fused with T18 or T25 subparts and have to be expressed at the external membrane of the bacteria. To ensure this,
 
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microscopy observations have been done with a DIBO group coupled with a fluorescent molecule : FITC. <br>
 
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'''Results of the COMP, COMP-T18 and COMP-T25 proteins marked show a great protein expression on the external membrane. (Link vers les résultats).'''<br>
 
 
<br>
 
<br>
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The bioluminescence intensity produced by the NanoLuciferase enzyme is analyzed.<br>
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Several conditions are tested with 100µL, 25µL, 5µL and 1µL of bacteria at OD600nm = 0.6 : respectively 48E+06 CFU, 12E+06 CFU, 24E+05 CFU and 48E+04 CFU .<br>
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===<u>NeuroDrop Project - Outer-Membrane BACTH</u> (mBACTH) ===
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In addition, times of induction are tested <u>from 0 to 360 minutes with 30 minutes increments</u>.<br>
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<div style="text-align:justify;">
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Cultures of the different recombinant bacteria are incubated overnight at 18°C under shaking in order to induce an optimal COMPs proteins production [http://2015.igem.org/Team:TU_Eindhoven cf Team Eindhoven 2015].<br>
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The low temperature allows a native protein folding and membrane insertion to avoids as much as possible the formation of inclusion bodies.<br>
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<br>
 
<br>
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Then cultures are diluted at OD600nm = 0,4 and let to grow to OD 600nm = 0.6 before induction.<br>
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https://static.igem.org/mediawiki/parts/0/0b/BACTH_constructions.gif
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The induction is performed by addition of '''0,5 mM IPTG''' and '''2mM of ATP''' for different periods of time. Bacteria are incubated at '''37°C''' under shaking (180 rpm) to allow an optimal '''NanoLuciferase production'''.<br>
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<br>
 
<br>
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After induction, 1, 5, 25 or 100µL of bacteria are distributed in a 96 wells black NUNC plate (ThermoFisher) the Nano-Glo® Luciferase Assay assay from Promega® is performed [https://france.promega.com/products/reporter-assays-and-transfection/reporter-assays/nano_glo-luciferase-assay-system/?catNum=N1110 (More informations)] : <br>
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<br>[http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015] iGEM project’s aim was to develop a “universal membrane sensor platform for biosensors”.<br>  
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“Prepare the desired amount of reconstituted Nano-Glo® Luciferase Assay Reagent by combining one volume of Nano-Glo® Luciferase Assay Substrate with 50 volumes of Nano-Glo® Luciferase Assay Buffer.<br>
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This year, '''Team Grenoble-Alpes''' is designing a new tears biosensor system based on [http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015]’s project.
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For example, if the experiment requires 10 mL of reagent, add 200μl of substrate to 10 mL of buffer.” The bioluminescence is then observed with a luminometer by measuring Relative Luminescence Units (RLU).<br>
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Both projects have a common base, the same receptors are used at the external surface of bacteria : '''[https://parts.igem.org/Part:BBa_K1492000 Clickable Outer Membrane Protein X(COMP)]'''. <br>
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<br>
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Several measures are made in the same well in order to reduce the incertitudes induced by the luminometer.<br>
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In order to test the '''reproducibility''' of our measures the means of '''3 differents experiments''' with '''3 measurements per well''' are measured with the calculation of standard deviation for each. <br>
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<br>
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'''Several controls are performed''':<br>
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'''∅ IPTG, ∅ ATP''' : To check the promoter leakage without any induction.<br>
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'''∅ IPTG, 2 mM ATP''' :To check if the addition of extracellular ATP helps the production of cAMP and to check if addition of ATP modifies the promoter leakage.<br>
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'''0,5 mM IPTG, ∅ ATP''' : To check if adding extracellular ATP is needed for protein expression.<br>
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'''0,5 mM IPTG, 2 ATP''' : Is the normal condition, it correspond to the measure at 360min.<br>
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<br>
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==Results==
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<br>
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−
The '''mBACTH''' following results are obtained with '''5µL''' of bacteria at OD600nm = 0.6 : '''24E+05 CFU'''.<br>
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<i>With 1µL (48E+04 CFU), the bioluminescence intensity was too low and the measurement were not discriminant enough.<br>
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Above 25µL of bacteria (12E+06 CFU), the signal was quickly saturated when the induction time increased and the luminometer could not give workable measures.<br>
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5µL (24E+05 CFU) is a good compromise, it’s enough to have a discriminant signal and sensitive enough to work as a small drop in our NeuroDrop system.<br>
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iGEM Grenoble-Alpes device NeuroDrop is designed for the use of small volumes like drops.
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Proving that 5µL of bacteria are enough to detect a significant difference in bioluminescence intensity between negative and positive condition is an encouraging result.
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Other reagents (see the full system) will be added to the drop of bacteria and its volume should not exceed 20µL for the engineers machine to work. 5µL of bacteria seem to be appropriate.</i><br>
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−
<br>
+
−
https://2019.igem.org/wiki/images/7/77/T--Grenoble-Alpes--mBACTH_Table_1.png <br>
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<i> Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the mBACTH generated with either <br>
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−
BBa_K3128018 and BBa_K3128017 : '''free sub-parts : negative condition''',<br>
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−
or BBa_K3128026 and BBa_K3128027 : '''Leucine Zipper : positive condition'''.<br>
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Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.</i><br>
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−
<br>
+
−
With '''1,48E+06 RLU''' of bioluminesce produced in the '''0,5 mM IPTG''' condition compared to '''9,02E+05''' in the condition '''without IPTG and without ATP''', it seems that <u>IPTG increase slightly the transcription</u>.<br>
+
−
Additionally, with '''2,55E+0,6 RLU''' of bioluminescence produced in the''' without ATP and 2mM ATP''' condition compared to '''9,02E+05''' in the '''without ATP and IPTG condition''', it seems that <u>ATP have a '''significant*''' effect on transcription</u>. <br>
+
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This was expected because of the lack of ATP in the periplasm of the bacteria. Thereby, <u>adding a great amount of ATP in the medium able to diffuse in the periplasm help the cAMP production by the periplasmic adenylate cyclase</u>.<br>
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Obviously, those observations don’t prove anything but give clues on the way the system operates. <br>
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<i>* A T test was done for the values of time above 90 min and led to a p-value below 0.01.</i><br>
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<br>
+
−
https://2019.igem.org/wiki/images/thumb/7/72/T--Grenoble-Alpes--mBACTH_Graph_1.png/800px-T--Grenoble-Alpes--mBACTH_Graph_1.png <br>
+
−
<i>Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the mBACTH generated with either <br>
+
−
BBa_K3128018 and BBa_K3128017 : '''free sub-parts : negative condition''', <br>
+
−
or BBa_K3128026 and BBa_K3128027 : '''Leucine Zipper : positive condition'''. <br>
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−
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.<br></i>
+
−
<i>* A T test was done for the values of time above 210 min and led to a p-value below 0.05.</i><br>
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−
<br>
+
−
<u>From 0 to 120 minutes</u> of induction time, the <u>bioluminescence produced by the two strains is similar</u>. <br>
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−
At '''120 minutes''', the <u>two curves start to split</u> and give rise to a <u>significant difference</u> between the free sub-parts : negative condition and the Leucine Zipper: positive condition from around '''210 minutes'''.<br>
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−
<br>
+
−
The discrepancy keeps increasing upon induction time, thus highlighting the efficiency of the '''amplification signal''' thanks to the signalling cascade and the strong reporter gene.<br>
+
  
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<u>From 0 to 120 minutes</u> of induction time, the <u>bioluminescence produced by the two strains is similar</u>. <br>
+
OmpX is an outer membrane protein with the C- and N-termini in the intracellular domain. To be able to use OmpX as a scaffold, a unnatural amino acid needs to be introduced.
−
At '''120 minutes''', the <u>two curves start to split</u> and give rise to a <u>significant difference</u> between the free sub-parts (negative condition) and the Leucine Zipper (positive condition) from around '''210 minutes'''.<br>
+
This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in,
−
<br>
+
which can be used for the SPAAC click chemistry reaction using DIBO functionalized groups, this modified protein is called '''COMP'''.
−
The discrepancy keeps increasing upon induction time, thus highlighting the efficiency of the '''amplification signal''' thanks to the signalling cascade and the strong reporter gene.<br>
+
The complex '''aptamer''' fixed to a '''COMP''' is then named a '''COMB''' for '''Clickable Outer Membrane Biosensor'''.<br>
 +
 
 +
<br>The Grenoble-Alpes team aims to develop an '''Outer membrane Bacterial Adenylate Cyclase Two Hybrid''' <i>(mBACTH)</i>.<br>  
 +
In this case, the two adenylate cyclase sub-parts are fused to the C-terminal ends of '''COMPs''' with a [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128010 Gly-Gly-Ser Linker (GGS)] of 54 amino acids <i>- in order to ensure a sufficient flexibility -</i>.<br>  
 +
When '''COMBs''' catch the extracellular target, they get closer, thus allowing the reconstitution of a functional adenylate cyclase due to the physical proximity of
 +
the two sub-parts.<br>  
 +
The enzyme is operational again and produce a high quantity of '''cAMP''' <i>(around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute)</i>,
 +
<u>the molecule responsible for the signal transduction in the bacteria</u>.<br>
 +
https://static.igem.org/mediawiki/parts/e/ec/BACTH_1.gif <br>
 +
 
 +
<br>'''cAMP''' molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP) in a ratio 1 to 1.
 +
Yhen two '''cAMP-CA'''P complexes are needed to activate the <u>expression of the gene under the control of the lactose promoter</u>.<br>
 +
Because of the high quantity of cAMP diffusing in the cytoplasm of the bacterium, the reporter gene is continously activated as long as cAMP is produced.<br>
 +
https://static.igem.org/mediawiki/parts/f/fd/BACTH_2.gif <br>
 +
 
 +
<br>The high enzymatic activity of <i>Bordetella pertussis</i> Adenylate Cyclase generates a high production of '''cAMP''' in presence of ATP in the bacterium
 +
thus <u>activating the signalling cascade with the CAP-cAMP dependant promoter</u>.<br>
 +
Hence this system is promising because it might have a great sensitivity and may drive a great signal amplification for a low amount of melocules to be detected.<br>
 +
</div>
  
 
<br>
 
<br>
−
==Conclusion==
 
−
'''There is a <u>significant difference between the negative and the positive condition of the mBACTH assay</u>''', <br>
 
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'''suggesting that a Bacterial Adenylate Cyclase Two-Hybrid can be successfully performed in the periplasm of bacteria which property is required for the sensing and detection of extracellular molecules.'''<br>
 
  
−
==User Reviews==
+
==References==
−
<!-- DON'T DELETE --><partinfo>BBa_K3128017 StartReviews</partinfo>
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<i>
−
<!-- Template for a user review
+
<sup>(1)</sup> Fields S, Song O. A novel genetic system to detect protein–protein interactions. Nature [Internet]. 1989<br>
−
{|width='80%' style='border:1px solid gray'
+
<sup>(2)</sup> Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. PNAS [Internet]. 1998<br>
−
|-
+
<sup>(3)</sup> Karimova G, Gauliard E, Davi M, P.Ouellette S, Ladant D. Protein–Protein Interaction: Bacterial Two-Hybrid. 2017<br>
−
|width='10%'|
+
Picture of the reaction ATP-cAMP. Khan Academy Website. Retrieved October 10, 2019, from https://www.khanacademy.org
−
<partinfo>BBa_K3128017 AddReview number</partinfo>
+
Euromedex, BACTH System Kit available here <br>
−
<I>Username</I>
+
Leusch, Paulaitis, Friedman. Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization. Am Soc Microbiol | Infect Immun. (1990) <br>
−
|width='60%' valign='top'|
+
Hantke, Winkler, Schultz. Escherichia coli exports cyclic AMP via TolC. J Bacteriol. (2011) <br>
−
Enter the review inofrmation here.
+
Eindhoven 2015 Website, from http://2015.igem.org/Team:TU_Eindhoven/Project/Design <br>
−
|};
+
</i>
−
<!-- End of the user review template -->
+
−
<!-- DON'T DELETE --><partinfo>BBa_K3128017 EndReviews</partinfo>
+

Latest revision as of 22:12, 21 October 2019

OmpX-WT protein fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter

Sequence and features

This biobrick is composed of the external membrane Outer Membrane Protein X Wild Type (OmpX-WT) protein fused at its C-terminal end to a 54 aa Gly-Gly-Ser Linker (GGS) followed by the T18 sub-part of the Bordetella Pertussis adenylate cyclase (AC) .
This biobrick is under a constitutive promoter J23101, thereby OmpX-WT fused to T18 protein is constitutively expressed leading to a large amount of recombinant protein in the outer membrane at any time.


This BioBrick is intended to operate with BBa_K3128018.
These two biobricks are the negative condition of the mBACTH -free sub-parts condition-.
OmpX proteins are fused to the adenylate cyclase sub-parts at their C-terminal ends. The fusion protein move freely in the bacterial outer membrane, but they are not forced to get closer by any mean.
The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.
The signal measured is considered as background noise.



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1339
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 880
    Illegal NgoMIV site found at 1290
    Illegal AgeI site found at 1096
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

In 1989, Fields and Song demonstrated a new genetic system allowing the detection of protein-protein interaction (1). At first, it was performed in Saccharomyces cerevisiae yeast and it was named the yeast two-hybrid assay (Y2H). In 1998, Ladant and al. described the system in bacteria (2). Nowadays, this biological technique is mostly used to show and characterize the physical interaction between two cytosolic proteins or internal membrane proteins in vivo (3).


Bacterial Adenylate Cyclase Two-Hybrid (BACTH)


The principle lies on the interaction-mediated reconstitution of a signalling cascade in Escherichia coli. The messenger molecule involved in this cascade is the cyclic adenosine monophosphate (cAMP) produced by the adenylate cyclase. Adenylate cyclase is an enzyme catalysing the cAMP production from ATP. It physiologically participates to the cellular transmission.


This system involves the Bordetella pertussis adenylate cyclase which is the responsible agent for the pertussis disease. Adenylate cyclase catalytic domain has the particularity to be splittable in two distinct parts: T18 and T25 sub-parts, unable to fonction unless they reassociate. Each sub-part of the enzyme is fused with a protein of interest, either the bait or the prey protein chose beforehand by the experimentator.

T--Grenoble-Alpes--BACTH_classicBACTH.gif


If two proteins interact, then T18 and T25 are bring together and reconstitute a functional adenylate cyclase enzyme thus enabling cAMP production. Using cya- bacteria – strain for whom the adenylate gene is deleted, involving an absence of this endogenous enzyme – a BACTH could be done with the creation of two fusion proteins : the first one, fused at its N or C terminal intracellular end with the T18 sub-part; the second one fused with the T25 sub-part.
The interaction of these proteins of interest will lead to the adenylate cyclase reconstitution, thus initiating cAMP production. The cAMP produced will act as a messenger by fixing itself to the transcriptional activator CAP, cAMP form the CAP-cAMP complex, controlling the expression of the lactose promoter by initiating transcription of the following gene.
This promoter is placed upstream the chosen reporter gene.


NeuroDrop Project - Outer-Membrane BACTH (mBACTH)


BACTH_constructions.gif

[http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015] iGEM project’s aim was to develop a “universal membrane sensor platform for biosensors”.
This year, Team Grenoble-Alpes is designing a new tears biosensor system based on [http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015]’s project. Both projects have a common base, the same receptors are used at the external surface of bacteria : Clickable Outer Membrane Protein X(COMP).

OmpX is an outer membrane protein with the C- and N-termini in the intracellular domain. To be able to use OmpX as a scaffold, a unnatural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction using DIBO functionalized groups, this modified protein is called COMP. The complex aptamer fixed to a COMP is then named a COMB for Clickable Outer Membrane Biosensor.


The Grenoble-Alpes team aims to develop an Outer membrane Bacterial Adenylate Cyclase Two Hybrid (mBACTH).
In this case, the two adenylate cyclase sub-parts are fused to the C-terminal ends of COMPs with a Gly-Gly-Ser Linker (GGS) of 54 amino acids - in order to ensure a sufficient flexibility -.
When COMBs catch the extracellular target, they get closer, thus allowing the reconstitution of a functional adenylate cyclase due to the physical proximity of the two sub-parts.
The enzyme is operational again and produce a high quantity of cAMP (around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute), the molecule responsible for the signal transduction in the bacteria.
BACTH_1.gif


cAMP molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP) in a ratio 1 to 1. Yhen two cAMP-CAP complexes are needed to activate the expression of the gene under the control of the lactose promoter.
Because of the high quantity of cAMP diffusing in the cytoplasm of the bacterium, the reporter gene is continously activated as long as cAMP is produced.
BACTH_2.gif


The high enzymatic activity of Bordetella pertussis Adenylate Cyclase generates a high production of cAMP in presence of ATP in the bacterium thus activating the signalling cascade with the CAP-cAMP dependant promoter.
Hence this system is promising because it might have a great sensitivity and may drive a great signal amplification for a low amount of melocules to be detected.


References

(1) Fields S, Song O. A novel genetic system to detect protein–protein interactions. Nature [Internet]. 1989
(2) Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. PNAS [Internet]. 1998
(3) Karimova G, Gauliard E, Davi M, P.Ouellette S, Ladant D. Protein–Protein Interaction: Bacterial Two-Hybrid. 2017
Picture of the reaction ATP-cAMP. Khan Academy Website. Retrieved October 10, 2019, from https://www.khanacademy.org Euromedex, BACTH System Kit available here
Leusch, Paulaitis, Friedman. Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization. Am Soc Microbiol | Infect Immun. (1990)
Hantke, Winkler, Schultz. Escherichia coli exports cyclic AMP via TolC. J Bacteriol. (2011)
Eindhoven 2015 Website, from http://2015.igem.org/Team:TU_Eindhoven/Project/Design