Difference between revisions of "Part:BBa K1638004"

Line 1: Line 1:
=Team Grenoble-Alpes 2019=
+
<partinfo>BBa_K1638004 short</partinfo>
==IMPROVE OF THE BACTERIAL ADENYLATE CYCLASE TWO HYBRID==
+
 
===Purpose===
+
This part codes for the T18 domain of the catalytic domain of the adenylate cyclase, CyaA, from Bordetella pertussis. When associated with the T18 domain of the catalytic domain of CyaA, the two domains together become active and catalyzes the conversion of ATP to cAMP. The rise in cAMP can in turn be used to trigger the expression of certain genes by using a cAMP-induced promoter.
The goal here is to prove that a <u>'''membrane''' bacterial two hybrid</u> ('''m'''BATCH) can be generated and can be functional by improving two biobricks from the original BACTH system:<br>
+
 
[https://parts.igem.org/Part:BBa_K1638004 BBa_K1638004] : T18 domain of adenylate cyclase from Bordetella pertussis<br>
+
The T25 domain can be found here: <partinfo>BBa_K1638002</partinfo>
[https://parts.igem.org/Part:BBa_K1638002 BBa_K1638002] : T25 domain of adenylate cyclase from Bordetella pertussis'''<br>
+
 
 +
<partinfo>BBa_K1638004 SequenceAndFeatures</partinfo>
 +
 
 
<br>
 
<br>
In order to do this, 4 biobricks are created:<br>
 
[https://parts.igem.org/Part:BBa_K3128017 K3128017] : '''OmpX Wild-Type''' (WT) protein fused with '''T18''' subpart of Bordetella Pertussis AC under constitutive promoter<br>
 
[https://parts.igem.org/Part:BBa_K3128018 K3128018] : '''OmpX WT''' protein fused with '''T25''' subpart of Bordetella Pertussis AC under constitutive promoter<br>
 
These two biobricks constitute the negative condition of the mBACTH (free sub-parts condition). OmpX proteins are fused to the adenylate cyclase sub-parts at their N-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 here is '''considered as background noise'''.<br>
 
<br>
 
[https://parts.igem.org/Part:BBa_K3128026 K3128026] : '''OmpX WT''' protein fused with '''[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128021 Leucine-zipper]''' (LZ) and '''T18''' sub-part of Bordetella Pertussis AC under constitutive promoter<br>
 
[https://parts.igem.org/Part:BBa_K3128027 K3128027] : '''OmpX WT''' protein fused with '''[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128021 Leucine-zipper]''' and '''T25''' sub-part of Bordetella Pertussis AC under constitutive promoter<br>
 
These two biobricks constitute the '''positive condition''' of the mBACTH (Leucine Zipper condition).
 
OmpX proteins are fused to the AC subparts at their N-terminal ends, and a leucine-zipper sequence is added between the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128050 signal peptide of OmpX] <i> -to express the recombiant protein in the external membrane- </i> and OmpX gene,
 
in order to force the physical closeness of OmpX proteins. <br>
 
Leucine zippers are peptides which contain a hydrophobic leucine residue at every seventh position.
 
They are able to dimerize through interactions between their helices.<br>
 
This is a strategy to '''force physical closeness'''. Hence the AC activity will be restored through the interaction of both subparts and will induce the cAMP dependant signalling cascade.<br>
 
<br>
 
The reporter gene used in the system is the NanoLuciferase enzyme present in the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128001 BBa_K3128001]
 
under a cAMP inducible [https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010 CAP-dependent lactose promoter].<br>
 
Two major factors affect this promoter :<br>
 
'''IPTG''', known to have a positive effect on the transcription of the gene by removing the lac repressor from the DNA.<br>
 
'''CAP''', known to have a positive effect on the transcription when it binds cAMP by helping the fixation of RNA-polymerase on the DNA<br>
 
To be able to bind the CAP sites on the promoter, the CAP protein has first to '''interact with a cAMP molecule'''. As soon as two cAMP-CAP complexes are bound to the CAP sites, the RNA Polymerase initiates the transcription.<br>
 
<br>https://2019.igem.org/wiki/images/thumb/7/79/T--Grenoble-Alpes--contribution-figure-1.png/800px-T--Grenoble-Alpes--contribution-figure-1.png <br>
 
<br>
 
ATP is not naturally present in large amount in the periplasm of the bacteria, thereby it has to be added in the bacteria medium to '''enhance''' its periplasm '''diffusion'''
 
and to be '''available''' for the adenylate cyclase catalytic reaction.
 
  
==='''Materials and Methods'''===
+
=Team Grenoble-Alpes 2019=
  
====Bacterial Strain====
+
<br>
<div style="text-align:justify;">
+
==Usage and Biology==
The assays are made with streptomycin resistant '''BTH101''' <i>E.Coli</i> strain, which are <i>cya-</i> bacteria.<br>
+
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>,
+
thus avoiding the presence of potential false positives and making the system more sensitive.
+
</div>
+
 
+
====Design of the plasmids====
+
 
<div style="text-align:justify;">   
 
<div style="text-align:justify;">   
To compare the efficiency of the BACTH system created with the initial '''BACTH''' biobricks '''BBa_K1638004 (containing the T18 subpart)'''  
+
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''  
and '''BBa_K1638002 (containing the T25 subpart)''', quantification results in BTH101 strain are needed.<br>
+
yeast and it was called 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
<i>pJT18</i> contains '''the T18 sub-part''' ; it has an ampicillin resistant gene and the pMB1 replication origin.<br>
+
used to show and characterize the physical interaction between two cytosolic proteins or internal membrane proteins in vivo <sup>(3)</sup>.  
<i>pJT18</i> contains [https://parts.igem.org/Part:BBa_K1638004 T18 subpart of Bordetella Pertussis AC] under constitutive promoter.<br>
+
<i>pJT25-Nlc</i> contains '''the T25 sub-part''' and the '''NanoLuciferase gene''' under the control of the '''plac promoter'''. It has a kanamycin resistant gene and the p15A replication origin.<br>  
+
<i>pOT25-Nlc</i> contains [https://parts.igem.org/Part:BBa_K3128001 NanoLuciferase reporter for BACTH assay] and  [https://parts.igem.org/Part:BBa_K1638002 T25 subpart of Bordetella Pertussis AC] under constitutive promoter.<br>
+
'''Those constructs will be the negative condition that show the background noise of the initial mBACTH system.'''<br>
+
 
<br>
 
<br>
<i>pJT18-ZIP</i> is similar to <i>pJT18-Nlc</i> with the addition of a '''Leucine Zipper''' sequence fused at the end of T18.<br>
 
<i>pJT18-ZIP</i> contains [https://parts.igem.org/Part:BBa_K1638004 T18 subpart of Bordetella Pertussis AC] fused with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128021 Leucine-zipper] under constitutive promoter.<br>
 
<i>pJT25-Nlc-ZIP</i> is similar to <i>pJT25-Nlc</i> with the addition of a '''Leucine Zipper''' sequence fused at the end of T25. <br>
 
<i>pJT25-Nlc-ZIP</i> contains [https://parts.igem.org/Part:BBa_K3128001 NanoLuciferase reporter for BACTH assay] and [https://parts.igem.org/Part:BBa_K1638002 T25 subpart of Bordetella Pertussis AC] fused with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128021 Leucine-zipper] under constitutive promoter.<br>
 
'''Those constructs will be the positive condition that show how the signal increases if both sub-parts are brought together with the BACTH.'''<br>
 
https://2019.igem.org/wiki/images/thumb/b/b9/T--Grenoble-Alpes--BACTH_Plasmide_1.png/800px-T--Grenoble-Alpes--BACTH_Plasmide_1.png<br>
 
<i>Genetic constructions of pJT18, pJT25-Nlc, pJT18-ZIP and pJT25-Nlc-ZIP plasmids used to test the cytoplasmic BACTH in BTH101 strain.</i><br>
 
  
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>
 
<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>
 
<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>
 
<i>pOT25</i> contains '''OmpX gene fused to the T25 subpart'''. It has a kanamycin resistant gene and the p15A replication origin.<br>
 
<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].<br>
 
'''Those constructs will be the negative condition that show the background noise of the initial mBACTH system.'''<br>
 
 
<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>
+
===<u>Bacterial Adenylate Cyclase Two-Hybrid</u> (BACTH)===
<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>
+
<div style="text-align:justify;">
<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>
+
<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>
+
'''Those constructs will be the positive condition that show how the signal increases if both sub-parts are brought together with the mBACTH.'''<br>
+
https://2019.igem.org/wiki/images/thumb/f/f9/T--Grenoble-Alpes--mBACTH_plamides.png/800px-T--Grenoble-Alpes--mBACTH_plamides.png<br>
+
<i>Genetic constructions of pOT18-Nlc, pOT25, pOT18-Nlc-ZIP and pOT25-ZIP plasmids used to test the membrane BACTH in BTH101 strain.</i>
+
</div>
+
  
===Transformation===
+
<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
<div style="text-align:justify;">
+
cyclic adenosine monophosphate (cAMP) produced by the adenylate cyclase. Adenylate cyclase is an enzyme catalysing the cAMP production from ATP. It
For the assay with the '''BACTH''', BTH101 are co-transformed either with <u><i>pJT18</i> and <i>pOT25-NlC</i> plasmids</u> : '''negative condition''',<br>  
+
physiologically participates to the cellular transmission. <br>
or <u><i>pJT18-ZIP</i> and <i>pJT25-Nlc-ZIP</i> plasmids</u> : '''positive condition'''.<br>
+
 
For the assay with the '''mBACTH''', BTH101 are co-transformed either with <u><i>pOT18-Nlc</i> and <i>pOT25</i> plasmids</u> : '''negative condition''',<br>  
+
<br>This system involves the <i>Bordetella pertussis</i> '''adenylate cyclase'''. You might know this bacterium; it is the responsible agent for the pertussis disease.
or <u><i>pOT18-Nlc-ZIP</i> and <i>pOT25-ZIP</i> plasmids</u> : '''positive condition'''.<br>
+
It adenylates cyclase catalytic domain has the particularity to be splittable in two distinct parts: '''T18''' and '''T25''' sub-parts, <u>unable to work 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>
 +
 
 +
<br>If the 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 promoter lactose by '''initiating transcription of the following gene'''. <br>
 +
This promoter is placed upstream from the chosen reporter gene.<br>
 
</div>
 
</div>
  
====Classic cytoplasmic BACTH and mBACTH assay====
 
<div style="text-align:justify;">
 
To test the two different BACTH systems, the bioluminescence intensity produced by the NanoLuciferase enzyme is analyzed.<br>
 
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>
 
In addition, times of induction are tested from 0 to 360 minutes with 30 minutes increments.<br>
 
 
<br>
 
<br>
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>
+
==IMPROVE==
The low temperature allows a native protein folding and membrane insertion to avoids as much as possible the formation of inclusion bodies.<br>
+
===<u>NeuroDrop Project - inter-Membrane BACTH</u> (mBACTH) ===
Then cultures are diluted at OD600nm = 0,4 and let to grow to OD 600nm = 0.6 before induction.<br>
+
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>
+
<br>
+
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>
+
“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.For example, if the experiment requires 10 mL of reagent, add 200μl of substrate to 10 mL of buffer.”<br>  
+
The bioluminescence is then observed with a luminometer by measuring Relative Luminescence Units (RLU).<br>
+
<br>
+
Several measures are made in the same well in order to reduce the incertitudes induced by the luminometer.<br>
+
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>
+
<br>
+
'''Several controls are performed''':<br>
+
'''∅ IPTG, ∅ ATP''' : To check the promoter leakage without any induction.<br>
+
'''∅ 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>
+
'''0,5 mM IPTG, ∅ ATP''' : To check if adding extracellular ATP is needed for protein expression.<br>
+
'''0,5 mM IPTG, 2 ATP''' : Is the normal condition, it correspond to the measure at 360min.<br>
+
<br>
+
</div>
+
===Results===
+
====Cytoplasmic BACTH assay====
+
 
<div style="text-align:justify;">
 
<div style="text-align:justify;">
The cytoplasmic BACTH, the following results are obtained with 5µL of bacteria (24E+05 CFU).<br>
 
https://2019.igem.org/wiki/images/3/32/T--Grenoble-Alpes--BACTH_Table_1.png <br>
 
<i>Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the BACTH generated with either <br>
 
BBa_K1638004 and BBa_K1638002 : '''free sub-parts : negative condition''', <br>
 
or BBa_K1638004 and BBa_K1638002 fused with BBa_K3128021 : '''Leucine Zipper : positive condition'''. <br>
 
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.<br></i>
 
 
<br>
 
<br>
With 7,48E+06 RLU of bioluminesce produced in the '''0,5 mM IPTG condition''' compared to 6,02E+06 in the condition '''without IPTG''' and 2mM ATP, it seems that '''<u>IPTG increase slightly the transcription</u>'''.<br>
+
https://static.igem.org/mediawiki/parts/0/0b/BACTH_constructions.gif
Additionally, with the <u>same produced bioluminescence<u> between the '''without IPTG and 2mM ATP''' and''' without IPTG and without ATP conditions''', '''<u>ATP appears to have no effect on transcription</u>'''.<br>
+
These two observations were expected because of the '''large amount of ATP already present in the cytoplasm of the bacteria saturating the adenylate cyclase'''. <br>
+
Obviously, those observations don’t prove anything but give clues on the way the system operates. <br>
+
 
<br>
 
<br>
https://2019.igem.org/wiki/images/thumb/b/b8/T--Grenoble-Alpes--BACTH_Graph_1.png/800px-T--Grenoble-Alpes--BACTH_Graph_1.png <br>
+
<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>  
<i>Luminescence production over time of induction for the '''negative condition strain (yellow curve)''' and the '''positive condition strain of the BACTH assay (purple curve)'''. <br>
+
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.
Area of the '''significant*''' difference between both curves is highlighted in yellow.<br>
+
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 called COMP]'''. <br>
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 90 min and led to a p-value below 0.05.</i><br>
+
  
This figure indicates that the production of luminescence by the two conditions are similar for the first 60 minutes of induction.  
+
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.
Then, a '''significant difference''' between the two strains is observed from '''90 minutes to 360 minutes''' of induction (yellow zone) with a continuous increase gap between both condition as time go on.
+
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,
This discrepancy highlights the efficiency of the expression of Nanoluciferase when the two subunits of the AC are forced to get closer as well as the amplification process of the system.<br>
+
which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups, this modified protein is called '''COMP'''.
 +
The complex '''aptamer''' fixed to a '''COMP''' is then called a '''COMB''' for '''Clickable Outer Membrane Biosensor'''.<br>
  
These data suggest that the classical cytoplasmic BACTH system is functional in BTH101 strain and can discriminate the presence or absence of the target from a <u>90 min induction</u>.<br>
+
<br>The Grenoble-Alpes team aims to develop an '''intermembrane Bacterial Adenylate Cyclase Two Hybrid''' <i>(mBACTH)</i>.<br>
</div>
+
In this case, the two adenylate cyclase sub-parts are fused to the N-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>
 +
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].<br>
 +
When '''COMPs''' and the aptamers 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 can produce the production of 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 of the signal transduction in the bacterium</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). One '''cAMP molecule''' binds to one transcriptional activator CAP;
 +
then 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 (2), the reporter gene is continually activated as long as cAMP is produced.<br>
 +
https://static.igem.org/mediawiki/parts/f/fd/BACTH_2.gif <br>
  
====Membrane BACTH assay====
+
<br>The high enzymatic activity (1) of <i>Bordetella pertussis</i> Adenylate Cyclase involves a high production of '''cAMP''' in presence of ATP in the bacterium
<div style="text-align:justify;">
+
thus <u>activating the signaling cascade with the CAP-cAMP dependant promoter</u>.<br>  
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.
+
Hence this system is promising because it might have a great sensitivity and may give a great signal amplification for a low amount of target detected.<br>
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.
+
COMPs are fused with T18 or T25 subparts and have to be expressed at the external membrane of the bacteria. To ensure this, microscopy observations have been done with a
+
'''DIBO group coupled with a fluorescent molecule : FITC'''.
+
Results of the <u>COMP, COMP-T18 and COMP-T25 proteins marked show a great protein expression on the external membrane</u>. (See results here).<br>
+
<br>
+
The mBACTH following results are obtained with 5µL of bacteria (24E+05 CFU).<br>
+
https://2019.igem.org/wiki/images/7/77/T--Grenoble-Alpes--mBACTH_Table_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>
+
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.</i><br>
+
<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>
+
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>  
+
Obviously, those observations don’t prove anything but give clues on the way the system operates. <br>
+
<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>
+
<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>Luminescence production over time of induction for the '''negative condition strain (red curve)''' and the '''positive condition strain of the mBACTH assay (blue curve)'''.<br>
+
Area of the '''significant*''' difference between both curves is highlighted in yellow. <br>
+
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.<br></i>
+
<br>
+
<u>From 0 to 120 minutes</u> of induction time, the <u>bioluminescence produced by the two strains is similar</u>. <br>
+
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>
+
<br>
+
Similarly to the BACTH system, 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>
+
 
</div>
 
</div>
  
====Comparison of the efficiency of the classic cytoplasmic BACTH and the external membrane BACTH of iGEM Grenoble-Alpes team.====
 
<div style="text-align:justify;">
 
https://2019.igem.org/wiki/images/thumb/a/a0/T--Grenoble-Alpes--mBACTH%2BBACTH_figure_1.png/800px-T--Grenoble-Alpes--mBACTH%2BBACTH_figure_1.png <br>
 
<i>Comparison of the efficiency of the classic cytoplasmic BACTH and the external membrane BACTH of iGEM Grenoble-Alpes team.  <br>
 
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.</i><br>
 
 
<br>
 
<br>
The results show a higher a quicker bioluminescence with the BACTH than with the mBACTH, this is due to multiple factors :<br>
 
The '''proteins diffuse more easily''' in the '''cytoplasm''' than in the '''outer membrane''' and so both sub-parts are more likely to encounter each other in the cytoplasm than in the outer membrane.
 
Whereof there is <u>more functional adenylate cyclase in the cytoplasm than in the outer membrane</u>.<br>
 
With traditional BACTH, '''cAMP is produced in the cytoplasm''' making it '''directly accessible''' to the proteins that '''enable transcription''' (CAP), unlike mBACTH that produce '''cAMP inside the periplasm'''.
 
Thereby, to be accessible by the CAP proteins cAMP need to diffuse in the cytoplasm thus <u>increasing the time needed to enable translation</u> and decreasing the quantity of cAMP reaching the
 
CAP protein therefore reducing the amount of NanoLuc produced.<br>
 
<br>
 
Nevertheless the '''mBACTH have a lower background''' noise thus allowing the discrimination of both condition.
 
At the end the '''mBACTH is operational''' if the bioluminescence detection and quantification is well optimise for our system.<br>
 
<br>
 
</div>
 
===Conclusion===
 
<div style="text-align:justify;">
 
'''Biobricks <u>BBa_K1638002</u> and <u>BBa_K1638004</u> have been improved in order to allow a functional bacterial two-hybrid system in the <u>bacterium periplasm</u>. <br>
 
The data show that OmpX protein is expressed at the external membrane of bacteria and that there is a <u>significant difference between the negative and the positive condition of the mBACTH assay</u>,
 
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.'''
 
</div>
 
  
==User Reviews==
+
==References==
<!-- DON'T DELETE --><partinfo>BBa_K1638004 StartReviews</partinfo>
+
<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%'|
+
</i>
<partinfo>BBa_K1638004 AddReview number</partinfo>
+
<I>Username</I>
+
|width='60%' valign='top'|
+
Enter the review inofrmation here.
+
|};
+
<!-- End of the user review template -->
+
<!-- DON'T DELETE --><partinfo>BBa_K312801 EndReviews</partinfo>
+

Revision as of 17:23, 11 October 2019

T18 domain of CyaA from Bordetella pertussis

This part codes for the T18 domain of the catalytic domain of the adenylate cyclase, CyaA, from Bordetella pertussis. When associated with the T18 domain of the catalytic domain of CyaA, the two domains together become active and catalyzes the conversion of ATP to cAMP. The rise in cAMP can in turn be used to trigger the expression of certain genes by using a cAMP-induced promoter.

The T25 domain can be found here: BBa_K1638002


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 550
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 91
    Illegal NgoMIV site found at 501
    Illegal AgeI site found at 307
  • 1000
    COMPATIBLE WITH RFC[1000]


Team Grenoble-Alpes 2019


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 called 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. You might know this bacterium; it is the responsible agent for the pertussis disease. It adenylates cyclase catalytic domain has the particularity to be splittable in two distinct parts: T18 and T25 sub-parts, unable to work 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.


If the 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 promoter lactose by initiating transcription of the following gene.
This promoter is placed upstream from the chosen reporter gene.


IMPROVE

NeuroDrop Project - inter-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 called 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 with DBCO functionalized groups, this modified protein is called COMP. The complex aptamer fixed to a COMP is then called a COMB for Clickable Outer Membrane Biosensor.


The Grenoble-Alpes team aims to develop an intermembrane Bacterial Adenylate Cyclase Two Hybrid (mBACTH).
In this case, the two adenylate cyclase sub-parts are fused to the N-terminal ends of COMPs with a Gly-Gly-Ser Linker (GGS) of 54 amino acids - in order to ensure a sufficient flexibility - followed by the T18 sub-part of the Bordetella Pertussis adenylate cyclase.
When COMPs and the aptamers 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 can produce the production of a high quantity of cAMP (around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute), the molecule responsible of the signal transduction in the bacterium.
BACTH_1.gif


cAMP molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP). One cAMP molecule binds to one transcriptional activator CAP; then 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 (2), the reporter gene is continually activated as long as cAMP is produced.
BACTH_2.gif


The high enzymatic activity (1) of Bordetella pertussis Adenylate Cyclase involves a high production of cAMP in presence of ATP in the bacterium thus activating the signaling cascade with the CAP-cAMP dependant promoter.
Hence this system is promising because it might have a great sensitivity and may give a great signal amplification for a low amount of target 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