Team:Chiba/Project/Delay Switch

Silencing the Speakers: Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
Desensitize Receivers: Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
Partial Blocking: Decreasing the by chewing the signal up.

Inter-species communications! We decided to go for the strategy inspired by Japanese-classic experience; Whenever we speak to somebody in English, we often experience a certain delay in activating the communication. We though this is exactly what we pursued in. See Exp #4. Same applies to the reverse, too. When somebody speaks to us, we definitely need some time (sometime infinite) to get activated. This is all in spite that he/ she was loud and clear enough. The less affinity (perception) we have to English, the longer we need to activate them.See exp #5.

Experiments (2008)

For details, click each of the index titles

Exp #1 Partial quenching of signals (jamming)

The idea was that the effective concentration of AHL in receiver cell get significantly decreased due to the constantly expressing AiiA, thereby enlarging the delay time.

Exp #2 Balancing Player

In this section, we intended to create time-delay by altering the amount of luxR protein per cell. Since the amount of LuxR that receives AHL differs, we hypothesized that the time required to reach the threshold AHL concentration will differ.

Exp #3 LuxR Mutants

Probably, the most straightforward approach to make variations in delay time is to create a number of LuxR mutants with different sensitivity. We thought the higher the sensitivity is, the shorter the delay time would be. The lower the sensitivity is, the slower the switch response should be.

Exp #4 Spoken to by Foreigners

In this plan, we induce cross-talk of quorum sensing by changing sender protein. This results in slower activation of receivers, when AHL concentration is increasing.

Exp #5 Speaking to Foreigners

In this plan, we induce cross-talk of quorum sensing by changing receiver protein. It is known that receiver proteins can work with the stimuli of signaling molecule even from another species of bacteria.

-> go to Team Chiba 2008 Project Page

Results (2008)

In conclusion, we tried (and are trying) to device a series of delay-switches by designing the "switching consortia". We got limited, by certain success in generating delay switches.

-> get more informations

Experiments (2009)

Exp #3' LuxR Mutants (One More Plan : Direct Screening for Delay Switcher)

In this plan, we introduced the random mutation into the entire reading frame of LuxR using error-prone PCR.

The mutants plasmid would be transformed with LuxP-gfp plasmid into E coli. Colonies were lifted onto nitrocellulose filter and placed on the top of lawn of Lux-senders. Over time, we kept watching the colony hue. Colonies should turn green when they detect the message passing through the NC filter. We were looking for the colony that turns green significantly after those of wildtype.

@@ Line 91: / Line 91: @@
 ==== [https://2008.igem.org/Team:Chiba/Experiments:copy_number Exp #2 Balancing Player] ====
 :In this section, we intended to create time-delay by altering the amount of luxR protein per cell. Since the amount of LuxR that receives AHL differs, we hypothesized that the time required to reach the threshold AHL concentration will differ.
+==== [https://2008.igem.org/Team:Chiba/Experiments:LuxR_mutants Exp #3 LuxR Mutants] ====
+:Probably, the most straightforward approach to make variations in delay time is to create a number of LuxR mutants with different sensitivity. We thought the higher the sensitivity is, the shorter the delay time would be. The lower the sensitivity is, the slower the switch response should be.
 ==== [https://2008.igem.org/Team:Chiba/Project/Experiments:Sender_Crosstalk Exp #4 Spoken to by Foreigners]====
@@ Line 106: / Line 109: @@
 === Experiments (2009) ===
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-==== [[Team:Chiba/Project/Delay_Switch/Mutants|Exp #3 Lux Mutants]] ====
+==== Exp #3' LuxR Mutants (One More Plan : Direct Screening for Delay Switcher) ====
-:Probably, the most straightforward approach to make variations in delay time is to create a number of LuxR mutants with different sensitivity. We thought the higher the sensitivity is, the shorter the delay time would be. The lower the sensitivity is, the slower the switch response should be.
+:In this plan, we introduced the random mutation into the entire reading frame of LuxR using error-prone PCR.
+The mutants plasmid would be transformed with LuxP-gfp plasmid into E coli. Colonies were lifted onto nitrocellulose filter and placed on the top of lawn of Lux-senders. Over time, we kept watching the colony hue. Colonies should turn green when they detect the message passing through the NC filter. We were looking for the colony that turns green significantly after those of wildtype.
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 === Results (2009) ===