Tokyo/Assay

From 2007.igem.org

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= Preliminary assays =
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__NOTOC__
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<br>[[Tokyo/Works|Works top]]　　0.[[Tokyo/Hybrid promoter|Hybrid promoter]]　　1.[[Tokyo/Formulation |Formulation]]　　2.[[Tokyo/Assay |Assay1]]　　3.[[Tokyo/Simulation |Simulation]]　　4.[[Tokyo/Assay2 |Assay2]]　　5.[[Tokyo/Future works |Future works]]
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=1.Important assay =
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== OD correction ==
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==[[Tokyo/Hill function fitting |Hill function fitting ]] ==
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新しく作ったハイブリッドプロモーターに関してはデータがないので，実験によりパラメータを求める．
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===Date: ===
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ヒル関数をfittingさせることにより，n2,n3,K2,K3が求まる.
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2007/09/21
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We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined n2, n3, K2, and K3.
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===Purpose: ===
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<br>[[Image:expression6-1.JPG|300px|]]
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===Samples: ===
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<br>[[Image:expression6-2.JPG|340px|]]
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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導出に関してはfor more detail.
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== Wash ==
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== [[Tokyo/Assays for hybrid promoter|Assays for hybrid promoter]] ==
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===Date: ===
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These assays were done
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20070925
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<br>-to determine the characteristics of our hybrid promoter.
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===Purpose: ===
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<br>The data from here were used for computational analyses and simulations.
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===Samples: ===
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== Detailed AHL assay ==
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== [[Tokyo/Repression and activation check with LacI and AHL|Repression and activation check with LacI and AHL]] ==
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目的
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===Date: ===
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結果
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20070925
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== [[Tokyo/IPTG assay|IPTG assay]] ==
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===Purpose: ===
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目的
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===Samples: ===
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== New-AHL assay ==
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結果
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== [[Tokyo/Activation check by cell-produced AHL |Activation check by cell-produced AHL ]] ==
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目的
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===Date: ===
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結果
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20070926
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=2.Preliminary assays=
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===Purpose: ===
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'''We conducted preliminary assays to practice experiments and check if the materials and methods worked properly.'''  
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===Samples: ===
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<br>[[Tokyo/Preliminary assays |See more details]]
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== LacI hybrid promoter Check ==
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=== Date: ===
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2007/09/20, 21
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===Purpose 1:===
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To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
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===Purpose 2:===
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To obtain parameters of LacI hybrid promoter for computational simulation.
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===Samples: ===
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<br>1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
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<br>2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
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<br>3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
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<br>4. pTrc322 TetR + [LacI promoter – GFP]
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<br>5. pBR322 TetR + [LacI promoter – GFP]
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<br>'''Repressor LacI expression:'''
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<br>pTrc99A expresses LacI
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<br>pBR322 TetR does NOT express LacI
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<br>'''Antibiotics resistance:'''
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<br>pTrc99A gives ampicillin-resistance
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<br>pBR322 TetR gives ampicillin-resistance
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<br>[LacI hybrid promoter – GFP] gives kanamicin-resistance
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<br>[placQI – GFP] gives kanamicin-resistance
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===Procedure:===
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= Assays =
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== AHL assay for Lux-lac hybrid promoter ==
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===Date: ===
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2007/09/20
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===Purpose: ===
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<br>check if AHL activates lux-lac hybrid promoter
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<br>check if lacI represses lux-lac hybrid promoter
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===Samples: ===
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<br>hybrid promoter in pTrc99A
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<br>hybrod promoter in pBR322
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<br>luxR in pTrc99A
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<br>luxR --- AHL-dependent activation confirmed
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<br>placqi on promter-less GFP in DH5a (for pos. con.)
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<br>promoter-less GFP in DH5a (for neg. con.)
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===Procedure: ===
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<br>prepare overnight culture for each sample
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<br>make fresh culture
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<br>take 3 ul of the overinight culture into 3 ml of LB (Amp and/or Kan) in Falcon tubes.  
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<br>incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter)
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<br>add AHL & IPTG solution
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<br>[AHL]final (in 3 ml LB culture) = 10 nM
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<br>[IPTG]final (in 3 ml LB culture) = 1 mM
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<br>incubate for 2 to 3 hours
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<br>apply 150 ul of samples into 96-well plaste
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<br>FLA measurement
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===Result: ===
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===Conclusion: ===
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<br>AND gate by AHL & IPTG
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<br>Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG.
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== '''AHL assay – LacI hybrid promoter activation by endogenous AHL''' ==
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[[Image:luxlachybrid.jpg]]
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=== Date ===
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2007/10/18
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=== Purpose ===
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To check if worker E. coli (Sender) can produce enough AHL for our model to work by using different copy numbers of plasmids.
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=== Samples　===
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placI luxI on PSB1(high copy) and A4Δp(Sender 1)
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<br>placI luxI on PSB4(low copy) and A4Δp(Sender 2)
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<br>placI luxI on PBR322(low copy) and A4Δp(Sender 3)
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<br>(no promoter)tetR pBR322 and A4 Hybrid promoter(Receiver 1)
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<br>(no promoter)luxI pBR322 and A4Δp
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=== Procedure ===
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0:00 Start to prepare over night culture (1 tube for each)
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<br>12:00 Make fresh culture with 3 ul of the overnight culture in 3ml of LB + Amp + Kan (LBAK) (3 tubes for each)
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<br>  Incubation in a shaker until the observed OD (ODobs) reaches up to 0.2
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<br>  Equalize ODobs of all the samples by adding LB media.
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<br>15:00　 Centrifugation at 5000 rpm and wash out the LB media.
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<br>Add cells with A4Δｐ and each of the following to 2.4 ml of LBAK
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[[Image:AHL assay endogenous.jpg]]
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<br>The experiments tested yellow boxes, not gray ones.
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<br>The total volume of E. coli culture (A4Δｐ and senders) was always 400 uM.
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wash
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take 200 ml of culture in a Falcon tube into a eppendorf tube (1.6 or 2.0 ml tube)
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<br>centrifugation for 2 min at 9000g
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<br>discard the supernatant with a pippet
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<br>add 200 ml of PBS and dissolve the pellet
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<br>centrifugation for 2 min at 9000g
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<br>discard the supernatant with a pippet
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<br>add 200 ml of PBS and dissolve the pellet
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<br>mix well
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<br>apply 150 ml onto a well
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----
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<br>16:00 (incubate it until the OD obs. <0.20)
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<br>17:30 stop incubation => wash* => FLA analysis（When OD reached 0.8）
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=== Results ===
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[[Image:AHL_assay_endogenous_Results.jpg]]
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=== Conclusion ===
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Not only high copy number plasmid pSB1, also low copy number plasmid pSB4 and pBR produced enough AHL to activate the LacI hybrid promoter in other cells. Especially, pBR remarkably produced AHL in the present experiment.
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Latest revision as of 14:06, 23 October 2007


Works top　　0.Hybrid promoter　　1.Formulation　　2.Assay1　　3.Simulation　　4.Assay2　　5.Future works

1.Important assay

Hill function fitting

新しく作ったハイブリッドプロモーターに関してはデータがないので，実験によりパラメータを求める． ヒル関数をfittingさせることにより，n2,n3,K2,K3が求まる. We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined n2, n3, K2, and K3.
Expression6-1.JPG
Expression6-2.JPG

導出に関してはfor more detail.

Assays for hybrid promoter

These assays were done
-to determine the characteristics of our hybrid promoter.
The data from here were used for computational analyses and simulations.


Repression and activation check with LacI and AHL

目的

結果

IPTG assay

目的

結果

Activation check by cell-produced AHL

目的

結果

2.Preliminary assays

We conducted preliminary assays to practice experiments and check if the materials and methods worked properly.
See more details

Retrieved from "http://2007.igem.org/wiki/index.php/Tokyo/Assay"