Chiba/Quorum Sensing

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[[Image:chiba_logo.png|center]]
[[Image:chiba_logo.png|center]]
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[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]<br>
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[[Chiba|Home]]<br>
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<span style="font-size:120%;font-weight:bold;">[[Chiba/Introduction|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]</span><br>
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
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==Size Control==
==Size Control==
===Our Aim===
===Our Aim===
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[[Image:Chiba_proj_ahlconc.png|frame|'''Fig1.''' Controlling AHL diffusing area and the size of Bacteria Marimo.]]
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[[Image:Chiba_proj_ahlconc.png|frame|'''Fig. 15''' Controlling AHL diffusing area and the size of Bacteria Marimo.]]
Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands.  This final state is not the case of real marimos.  Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.  
Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands.  This final state is not the case of real marimos.  Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.  
#Raise the AHL productivity of Sender
#Raise the AHL productivity of Sender
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AHL is synthesized from methionine by the enzyme MetK and LuxI.<br>
AHL is synthesized from methionine by the enzyme MetK and LuxI.<br>
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.
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<br>[[Image:Chiba_ahlsynth.png|frame|Fig.2 Passway synthesizing AHL in bacteria.]]
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<br>[[Image:Chiba_ahlsynth.png|frame|'''Fig. 16''' Passway synthesizing AHL in bacteria.]]
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===Experiment===
===Experiment===
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[[Image:Chiba AHL test 02.jpg|frame|fig3. Scheme of AHL test.]]
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[[Image:Chiba AHL test 02.jpg|frame|'''Fig. 17''' Testing procedure of sender capability.]]
====Sender====
====Sender====
Ptet-LuxI
Ptet-LuxI
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====metK Sender====
====metK Sender====
[[Image:metK circuit.jpg]]
[[Image:metK circuit.jpg]]
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*Synthesize AHL and express metK constantly
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*Synthesize AHL / express metK (precursor supplier) constantly
====Receiver====
====Receiver====
[[Image:BBa T9002.jpg|350x48px]]
[[Image:BBa T9002.jpg|350x48px]]
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*BBa T9002 (Express GFP in response to AHL)
====Method(Fig3.)====
====Method(Fig3.)====
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#Inoculated sender, MetK sender, and receiver in each liquid medias.  Incubated at 37℃ 12h.
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#Transform sender (Ptet-luxI) and MetK sender (pLac-luxI-metK) into E coli strains (XL10Gold and BW25113)
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#Checked OD<sub>600</sub>.  Dispensed receiver in each tube equally.  Spin downed senders and resuspended with fresh liquid media.
+
#Inoculated them independently in liquid media.  Incubated at 37℃ 12h.
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#Diluted senders to adjust cell population (5x10<sup>8</sup>,5x10<sup>7</sup>,......, 5x10<sup>2</sup>).
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#Checked OD<sub>600</sub>, washed, and diluted senders to adjust cell number (5x10<sup>8</sup>,5x10<sup>7</sup>,......, 5x10<sup>2</sup>).
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#Mix receivers and senders.
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#Mix with receiver (pLux-GFP/ BW25113, 1.5x10<sup>9</sup> cells)
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#Incubated for 1h to 3h at room tempature
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#Shake for 1-3h at room tempature (optonal; methionine (10mM) added)
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#Spindowned and UV checked.
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#Spindowned and fluorescence checked.
 +
===Result===
 +
[[Image:AHL test photo 01.jpg|frame|left|'''Fig. 18''' Testing minimum amount of senders required for activating receivers.<br> ]]
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*At least 5x10<sup>4</sup> cell/ml sender required to activate lux promoter in BW25113.
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===Result===
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*Sender performance HIGHLY depends on the strains: 100x more cells were required to activate the lux promoter in other strain XL10Gold.
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[[Image:AHL test photo 01.jpg|frame|left|'''Fig. Methionine 0mM.'''<br> metK(+): metK Sender ( pLac-luxI-metK )<br> metK(-): Sender ( Ptet-luxI )]]
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*Overexpression of metK did not result in the booster effect. It was so even when Methionine (10mM), the direct substrate of metK, was added to the growth media (not shown). Sucks.
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[[Image:AHL test photo 02.jpg|frame|left|'''Fig. Methionine 10mM.''' metK(+): metK Sender ( pLac-luxI-metK )<br> metK(-): Sender ( Ptet-luxI )]]<br clear="all">
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*At least 5x10<sup>4</sup> cell/ml sender required to activate lux promoter.
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*Sender performance HIGHLY depends on the strains.  
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*No boosting effect was observed by overexpression of metK gene. It was so even when Methionine, the direct substrate of metK, was added to the growth media. Sucks.
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===Discussion===
===Discussion===
*No improbement in sending power was observed.  
*No improbement in sending power was observed.  
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*Assumption (not conclusion): (1) Suply of S-adenosyl methionine (precursor) is probably enough, and luxR is by far the rate-limiting step in AHL synthetic pathway. (2) There exist control mechanism of AHL precursor; overexpression of metK ativates the degradation pathway of its product. Need more reading.
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*Assumption (not conclusion):<br>
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*Note; We haven't confirmed the entire sequencing of the metK gene (PCR cloned) used in this experiment.
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(1) Precursor supply (S-adenosyl methionine) is simply at enough level, and luxR is by far the rate-limiting step in AHL synthetic pathway. No chance/ need for metabolic engineering of this pathway. <br>
 +
(2) Precursor flows out to somewhere else. Or overexpression of metK ativates the degradation pathway of its product. Anyway too hard for us to cope with in short time.
 +
<br>
 +
*Note; We haven't confirmed the entire sequencing of the metK.
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==2.Improving Receiver==
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==2.Sensitizing Receiver==
===Design===
===Design===
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[[Image:MLuxRx.gif|frame|left|Fig. Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)]][[Image:MLuxRxxx.gif|frame|left|Fig.Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)]]<br clear="all">
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[[Image:MLuxRx.gif|frame|left|'''Fig. 19''' Transfer curve of LuxR mutant to AHL.''' Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)''']][[Image:MLuxRxxx.gif|frame|left|'''Fig. 20''' LuxR mutations focused in our project ]]<br clear="all">
Collins ''et.al.'' described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. ''Mol. Microbiol.'' '''55''', 712–723 (2005))<br>
Collins ''et.al.'' described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. ''Mol. Microbiol.'' '''55''', 712–723 (2005))<br>
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We created two types of sensitive luxR mutants.<br>Because there is no data shows 2point(I45F,S116A) mutated luxR.We expected something new data of mutation will be found. Also we want to obtain sensitive(not hyper sensitive) receiver.<br>
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We created two luxR mutants. One is I14F mutant of LuxR, and another is I45F/S116A.<br>  
 +
There is no report on I45F/S116A double mutant.By creating/ analyzing this mutant in AHL sensitivity, we can tell the effect of T33A in LuxR.<br>
===Experiment===
===Experiment===
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[[Image:MLuxRxx.gif]]<br>
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[[Image:MLuxRxx.gif|frame|'''Fig. 21''' Location of the mutations in luxR gene]]<br>
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Sensitive luxR mutants assessed by follow method.<br>
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luxR mutants was analyzed in their sensitivity to AHL by following procedure.<br>
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1.Inoculate Receiver(wild type luxR/BW25113), mutated Receiver(1point mutation/BW25113) in liquid media 37℃ 12hour.
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#Inoculate Receiver (wild type luxR/BW25113), mutated Receiver (1point mutation/BW25113) in liquid media for 12 h at 37℃.
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2.Despence them equally.
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#Adjust the cell density and add AHL (final concentration, 0, 0.1, 1, 5, 10, 100, and 1000 nM)
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3.Add AHL final concentration(1μM,100nM,10nM,5nM,1nM,0.1nM)
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#Incubate for 1h at room temperature
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4.1hour at room temperature
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#Spindown cells and GFP folurescent level checked<br><br><br>
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5.Spindown and UV checked.
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===Result===
===Result===
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[[Image:MLuxR-test.gif|frame|left|Fig.comparison 1point mutated and wild type]]
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[[Image:MLuxR-test.gif|frame|left|'''Fig. 22''' comparison 1point mutated and wild type]]
*Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.  
*Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.  
<br><br><br><br><br><br><br><br><br>
<br><br><br><br><br><br><br><br><br>
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[[Image:Two mutation.jpg|320x240px|frame|left|Fig.2point mutation(without AHL)]]
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[[Image:Two mutation.jpg|320x240px|frame|left|'''Fig. 23''' LuxR mutant with two point mutations expressed GFP without adding AHL]]
*2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.
*2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.
<br><br><br><br><br><br><br><br><br><br><br><br><br><br>
<br><br><br><br><br><br><br><br><br><br><br><br><br><br>
===Discussion===
===Discussion===
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[[Image:MluxR_table.jpg |frame|left|'''Table 1. Seisitivity of LuxR mutants.''' Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)]]<br><br><br><br><br><br><br><br><br>
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[[Image:MluxR_table.jpg |frame|left|'''Table 1 Seisitivity of LuxR mutants.''' Data modified from Collins ''et.al.'' Mol. Microbiol. 55, 712–723 (2005)]]<br><br><br><br><br><br><br><br><br>
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GFP expression was not observed on the plate of aiiA receiver.
GFP expression was not observed on the plate of aiiA receiver.
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[[Image:T9002 plate.jpg|frame|Receiver|left]]
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[[Image:T9002 plate.jpg|frame|'''Fig. 24''' Receiver|left]]
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[[Image:AiiA cons plate.jpg|frame|aiiA receiver|center]]
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[[Image:AiiA cons plate.jpg|frame|'''Fig. 25''' aiiA receiver|center]]
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Latest revision as of 05:41, 27 October 2007

Chiba logo.png

Home
Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal
Acknowledgements | Team Members | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | メンバ連絡簿


Size Control

Our Aim

Fig. 15 Controlling AHL diffusing area and the size of Bacteria Marimo.

Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands. This final state is not the case of real marimos. Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.

  1. Raise the AHL productivity of Sender
  2. Increase the AHL sensitivity of Receiver
  3. Use the AHL degrading enzyme aiiA to localize AHL


1.Improving Sender

Design

AHL is synthesized from methionine by the enzyme MetK and LuxI.
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.


Fig. 16 Passway synthesizing AHL in bacteria.


Experiment

Fig. 17 Testing procedure of sender capability.

Sender

Ptet-LuxI

  • Synthesize AHL constantly

metK Sender

MetK circuit.jpg

  • Synthesize AHL / express metK (precursor supplier) constantly

Receiver

BBa T9002.jpg

  • BBa T9002 (Express GFP in response to AHL)

Method(Fig3.)

  1. Transform sender (Ptet-luxI) and MetK sender (pLac-luxI-metK) into E coli strains (XL10Gold and BW25113)
  2. Inoculated them independently in liquid media. Incubated at 37℃ 12h.
  3. Checked OD600, washed, and diluted senders to adjust cell number (5x108,5x107,......, 5x102).
  4. Mix with receiver (pLux-GFP/ BW25113, 1.5x109 cells)
  5. Shake for 1-3h at room tempature (optonal; methionine (10mM) added)
  6. Spindowned and fluorescence checked.

Result

Fig. 18 Testing minimum amount of senders required for activating receivers.


  • At least 5x104 cell/ml sender required to activate lux promoter in BW25113.
  • Sender performance HIGHLY depends on the strains: 100x more cells were required to activate the lux promoter in other strain XL10Gold.
  • Overexpression of metK did not result in the booster effect. It was so even when Methionine (10mM), the direct substrate of metK, was added to the growth media (not shown). Sucks.

Discussion

  • No improbement in sending power was observed.
  • Assumption (not conclusion):

(1) Precursor supply (S-adenosyl methionine) is simply at enough level, and luxR is by far the rate-limiting step in AHL synthetic pathway. No chance/ need for metabolic engineering of this pathway.
(2) Precursor flows out to somewhere else. Or overexpression of metK ativates the degradation pathway of its product. Anyway too hard for us to cope with in short time.

  • Note; We haven't confirmed the entire sequencing of the metK.

2.Sensitizing Receiver

Design

Fig. 19 Transfer curve of LuxR mutant to AHL. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)
Fig. 20 LuxR mutations focused in our project

Collins et.al. described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005))
We created two luxR mutants. One is I14F mutant of LuxR, and another is I45F/S116A.
There is no report on I45F/S116A double mutant.By creating/ analyzing this mutant in AHL sensitivity, we can tell the effect of T33A in LuxR.

Experiment

Fig. 21 Location of the mutations in luxR gene

luxR mutants was analyzed in their sensitivity to AHL by following procedure.

  1. Inoculate Receiver (wild type luxR/BW25113), mutated Receiver (1point mutation/BW25113) in liquid media for 12 h at 37℃.
  2. Adjust the cell density and add AHL (final concentration, 0, 0.1, 1, 5, 10, 100, and 1000 nM)
  3. Incubate for 1h at room temperature
  4. Spindown cells and GFP folurescent level checked


Result

Fig. 22 comparison 1point mutated and wild type
  • Both wild type and 1point mutation luxR express GFP under 5nM AHL concentration.










Fig. 23 LuxR mutant with two point mutations expressed GFP without adding AHL
  • 2point mutation(I45F,S116A) luxR Receiver express GFP without AHL.















Discussion

Table 1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)










This table is quoted from paper.
In the paper, DH5α strain was used.
But wild type luxR in our study expressed GFP under 5nM AHL concentration.

Assumption:There is some difference between strains.
Required to do same test on other strains

3.Localizing AHL

Design1

BBa I729006 circuit.jpg
BBa I729006
In addition to the receiver unit, there added AiiA under the control of lac promoter.
AiiA degrades/ inactivates AHL, lowering the AHL concentration in the cell.
This way cells installed with this circuit can respond, but less sensitive, to the sender cells.

Experiment

  1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
  2. At the same time, inoculate AHL sender E.coli in Liquid Media.
  3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
  4. Check plate.(GFP expressed?)

Result

GFP expression was not observed on the plate of aiiA receiver.

Fig. 24 Receiver
Fig. 25 aiiA receiver


Discussion

We thought expression of aiiA significantly decrease the cellular level of AHL. It turned out to be true.
However, it went too much. It does not respond to senders anymore, probably due to too fast AHL degradation.
We need more controlled/ moderate expression of aiiA.

Design2

Plux aiiA.jpg
This gene circuit is expected so that aiiA is synthesized only when the receiver senses AHL signal. If so, the whole Bacteria Marimo becomes AHL quencher. In fact, the bacteria inside of the Marimo degrades AHL and the AHL diffusion outside of Marimo is inhibited.

Experiment

  1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
  2. At the same time, inoculate AHL sender E.coli in Liquid Media.
  3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃ 12hour
  4. Put plate 30℃
  5. Check GFP expression every 1hour.

Result

  • When we compared the GFP expression with the test tube of BBa_T9002, the bacteria with the consructed gene circuit did not fluoresce immediately after incubation, however fluoresced in 18~20 hour.

Discussion

  • GFP expression means plux is activated. So we could make moderate aiiA receiver.
  • But we need more twisted circuit.

Design3

Rec inv aiia.jpg
Above picture describes aiiA is regulated inverted lux promoter with CI inverter.

  • High AHL concentration: no aiiA expression, no AHL degrade.
  • Low AHL concentration: aiiA expressed, AHL degraded.


Experiment

We could not finish assembling this part. It will be our future work.

Subpart:BBa_S03840

S03840.jpg