The Agrobacterium reporter strain is very fickle....so some steps would be to ensure that you culture has gone through these two steps

step1. fresh glycerol stock inoculated and kept overnight

step2. the culture from step 1 should be further inoculated and grown overnight

For TLC, spot your extract and run it on 6:4 Methanol:water,allow to dry and on this TLC plate pour a 0.5% agar media solution( with 1%of your culture inoculum from step2).An overnight inoculum will help you visualise your AHL-active spot

As for LCMS you can use a C18 column for seperation in an Acetonitrile(Acn) water gradient (The column is initially eluted with 10 % acetonitrile (ACN) at 1ml/min for 10 mins followed by a linear gradient to reach 100% ACN in 65 minutes and an additional 15 min at 100% ACN.)

In the generated MS data, check for m/z peak corresponding to 101/102(lactone fragment) or 143/144 (Mclafferty fragment) and the corresponding molecular ion peak

for example if your AHL is oxoC8HSL u will get m/z corresponding to 241/242 as your molecular ion peak. in addition to m/z 101/102 or 143/144 (or both)

HOPE THIS HELPS

Thanks Pramal I am following the same procedure with 0.7% agar, but it didn't work. Will try with 0.5%. Did u tried the same with Chromobacterium CV026??

Its me.

How do you carry out extraction step? What type of sample do you use? We need more specific experiment information for solving yout problem. The identification of streaking did not need extration sample, but TLC and LC need extraftion sample from biofilm. The extraction method was figure out on the web site.

Also, CV026 is only detected short chain homoserine lactone. If you use long chain standard HSL, you can not take result.

Hello Neelam

As a matter of fact i have used CV026. CV026 works in two ways

1.Direct detection for short chains C4,C6 AHL

2.Whereas for long chain and oxo containing AHLs you can use the inhibition assay.For this you have to mix your CV026, either of the Short chain AHLs and the 0.5% agar and pour them in a petri plate. then use a disc or a well to load your extract to the setup.A zone of clearance in the violet lawn the following day will suggest the presence of long chain or oxo AHLs.The same can be tried with TLC bioassay

P.s death of CV026 could be viewed as a false positive result .......avoid this by using extract concentration lower than the minimum inhibitory concentration

Hi Pramal....

1. For long chain or oxo containing AHLs instead of CV026, will parent chromobacterium work??

2.Are you sure that Cv026 work in other way to detect long AHLs or oxo AHLs??? because quorum quenching/antibacterial activity by some other compound in the crud extract may give the same results..

And thats where your Agrobacterium data will be of help as the same extract cannot be QS inducing and QS inhibiting.The reason why we use multiple QS reporters

Parent CV026 works with Oxo and long chain AHLs in the reverse way.I have personally used it for pure oxoC8 and OxoC12

Pramal, but the results for CV026 or parent chromo, with Oxo AHLs is due to AHLs signalling quenching or a true AHLs assay?..

I can't actually give you a 100% answer on that coz oxoAHLs induce QS activities in some bioreporter strain but in CV026 it inhibits the QS phenotype of violacein production......But idealy if your strain has activated some reporter strains and is producing a clearance zone in CV026 then it could be estimated to produce a long chain or oxo AHLs .......obviously the data requires further proof for AHLs presence like a valid GC or LCMS Spectra

Hello everybody! Hello everybody! I've the same big problem of Neelam with TLC for A.tumefaciens!

I tried with the TLC methods (that explained Pralam)...but which media you used? I've used Minimal media but didn't work!!! I tried with the colture (step 2 ) with OD 0,5 and 0,1. But nothing! anyone could help me?? please!

What kind of HSL do you used? Normally, Agrobacterium tumefaciens A136 [traI-lacZ fusion(pCF218)(pCF372)] detect C6HSL to C14-HSL by β-galactosidase activity. A. tumefaciens strain NT1 (pDCI41E33 containing a traG::lacZ fusion) detect AHLs with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths, (C6 ~ C14HSL) with the exception of C4HSL by β-galactosidase activity. And, C. violaceum CV026 (cviI::mini-Tn5) detect C4 ~ C8-HSL and 3-oxo-C4~C8-HSL by AHL production impaired. And, tell us specific concentration of standard HSL. Sometimes, HSL concenctration made problem of their phenotype. And, CV026 can be grow LB medium.

Sara i got best results with luria bertani media whose compostion( for 1Litre) is

Tryptone 10 g

Yeast Extract 5 g

NaCl 10 g

This media you can try with 1% or 0.5% Agar to get desired results.Hope It helps

Hi Kim and Pramal, thaks for the anwswer!

I used A.tumefaciens NTL4(pZLR4) that detect HSL C-12 by B galactosidase activity. I have used Minimal media for overlay the TLC plates (Minimal media added with X-gal)...and the concentration of the positive control(C-12) was 2µl, instead, I put 25µl of each sample (the extract).

Pramal do you use the LB agar with A.tumefaciens, whitout X gal???

I have used LB soft agar for S.marcescens and C.violaceum and worked, but the problem is A.tumefaciens: I get the whole TLC plates blue colored, and the spots relative at my samples not clear and white...instead would be the opposite!!!

What do you suggest me????

Even i have encountyered the same problem with Agrobacterium Sara.... i have used LB with Agrobacterium with Xgal .Here are a few points based on what i know

1.Try using 1% or 1.5% agar.They prevent spreading of the blue colour

2.At TLC level load just 5uL of your extract

3. if your checking for knowing the AHL producing capability of your extract.You can check the extract TLC and control TLC on seperate plates.

Please apply these techniques.It has worked for me

Ok Pramal of course, I'll try!!! Last quastion...for overlay, how much OD of A.tumefaciens have you added at LB media??

Normally I used to use 0,5 OD!

And about the concentration of X-gal, I put 400µl/400ml! That's it's right?

The blue color range depend on concentration of HSL. If you used very high conc of HSL, your plate was coverd fully blue color. In this case, you should decrese conc of HSL level. And, I am not sure about NTL4. Nomarlly, I used A136 for reporter strain. Good luck to you~. And, blue color range (or phenotype level) of reporter strain (A136 or NTL4 or everythings..) depend on type of HSL. In my case, blue color (of A136) is more stronger 3-oxo-C8 HSL than C8-HSL. If you want to compare of each HSL, you should controlled its conc..

its all a matter of trail and error ......i guess sara. I use initital 0.1 OD and grow it till 48hours in xgal supplemented media at 25oC and then observe

One more point incubate your culture at temperatures below 30 to get better results

Feel free to ask for more doubts.will try helping

Hello Pramal Biswa,

LCMS you can use a C18 column for seperation in an Acetonitrile(Acn) water gradient (The column is initially eluted with 10 % acetonitrile (ACN) at 1ml/min for 10 mins followed by a linear gradient to reach 100% ACN in 65 minutes and an additional 15 min at 100% ACN.)

In the generated MS data, check for m/z peak corresponding to 101/102(lactone fragment) or 143/144 (Mclafferty fragment) and the corresponding molecular ion peak.

May I know the reference for this protocol

Please what will be my "test organism or solution" when carrying out quorum sensing experiments? Edit

The quorum sensing is to be done using C. Violaceum CVO26 and A. Tumefaciens NTL4 reporter strains. I'm using the cross-feeding plate assay and inoculation strategies for bio-assays on petri plates. This is my first time carrying out quorum sensing experiments. I'll appreciate all the help I can get. Thanks.