I don't understand. Do you mean your plasmid doesn't function after ligating a piece of DNA into a multiple cloning site, or that creating a fusion protein is disrupting protein function, or something else?

Thank you for the query. We are trying to create a plasmid with the LUx operon and PDAB vector. The aim is to see bioluminescence upon adding arabinose (which triggers PBAD promoter) to the culture having the gene of interest (after transformation). Though we could generate the plasmid ( after ligation, doing a miniprep from the colonies on the ligation plate ) but cannot see bioluminescence upon adding arabinose (2mg/mL) to the solution. Look forward to your excellent guidance.

Do you have a positive control to check that your arabinose induction is working? If so, could you describe it and also post a map of your pBAD-LUx construct?

yes we have a positive control with only PGLOW which did glow under UV. During ligation we only had the two parts ligated together (Lux and PBAD) and transformed in E coli.Then two to three colonies were picked up and added to 4 mL of LB containing amp and arabinose and monitored for two days at room temperature, no bioluminescence observed. Here goes the map.

thank you!

Mithu- you probably already know this, but make sure no glucose is present in your culture medium, since it inhibits uptake of arabinose, and that you are using L-arabinose for induction. I haven't worked with the LUx operon, but wonder whether any regulatory sequences might be present at the 5' end that are serving to repress transcription. Your positive control relies on UV exposure- are you sure the regulatory elements responsible for this response are absent in the subcloned 11kb fragment?

thank you for the reply and excellent advice, i will definitely recheck whether any glucose is there or not. The positive control plasmid is as follows where we have replaced the GFP with the LUX operon expecting it to glow upon arabinose induction.

Look forward to your excellent comments!

Thanks for posting the pGLOW map. Thought UV was your inducer- now I understand more clearly. Arabinose induced GFP expression that you could detect under UV illumination, right? This indicates that you are able to get the pBAD promoter to activate.

In this case, it seems that the problem lies within the LUX operon. Make sure you sequence the insert. Also, the natural LUX operon is controlled by a separate autoinducer-receptor complex (R gene). This should be absent from your system, but I'm not completely sure based on your map. The cloning should start with the LuxC gene, and not contain other elements upstream of it.

What bacterial strain are you using?

Thank you for your thoughtful insight and excellent suggestion. We definitely want to sequence the insert.

Bioluminescence of Lux operon is regulated by quorum sensing (a particular high bacterial concentration) which is controlled by R gene and the I gene (which generates the signaling autoinducer for quorum sensing). The aim was to get rid of quorum sensing by changing the promoter (R and I genes) with the PBAD promoter such that addition of arabinose can generate bioluminecence.

Lux operon is a huge fragment and sequencing is truly a good idea.

HB101 is the bacterial strain.

Please let me know your valuable suggestions.

Thank you!

Mithu- I would also suggest trying to see if the quorum sensing works with the original LUX vector in the HB101s. Just to validate that you started with a functional operon and that nothing strange is going on with your host strain.

We are trying to troubleshoot what might have happened. The plasmid is made up of two parts, the LUX operon and the PBAD backbone. I suggested to cut out the Lux operon and put back the GFP in that place. If the PBAD part is same as that in PGLOW then the new GFP and PBAD backbone (ligated together) will glow under UV. From this experiment we can be sure that the PBAD part is OK then we can move ahead with the LUX part.

In this way we can save money and time to prevent sequencing of the entire PBAD and LUX plasmid.

The GFP primers are there and we have started the GFP amplification in PCR.