Suziebee

Hi guys,

I was wondering does anyone have a protocol/tips for transforming (electrotransformation) Campylobacter species? They have restriction endonucleases, CRISPR systems and may not recognise plasmids with E. coli ORI's. I've transformed many other bacterial speices with pretty much no problems however Campylobacter is proving very difficult.

Thanks in advance for any help/ advice/ tips!

All the best,
Susan

Hi,

Hopefully someone may be able to help me.  I currently conducting a trial using IL10-/- mice to evaluate the role of an emmerging bacterial gastrointestinal pathogen.
The gross pathology from our prelimary trials look promising, enlarged colon, enlarged spleen and diahorria however I would like to measure the immune response of the mice to this potential pathogen; for instance Thelper1 cells, Tregs, FoxP3.

My poblem is that I do not have access to a flow cytometer so I am hoping somebody may have ideas for an alternative to flow? I was thinking ELISPOT or ELISA but the companies cac't recomend them for tissue homogenate.

I would really appreciate any advice people may have!

Thanks in advance,
Susan

Hi guys,

I'm wondering could someone help me with interpreting my SDS-PAGE gel.

I am looking for the presence of a Capsular Ploysaccharide on my bacterium, so I ran a 12% SDS-PAGE with freshly harvested cells, washed twice with PBS, and resuspended in Saline and heated at 50°C for 1hour (CPS is thermostabile) and centrifuged. The supernatant was removed and boiled with sample buffer for 5mins and loaded onto the 12% sds-gel.

I stained the gel with alcian blue and have discrete bands around 70-100kDa. There is no mAb fro the CPS of this bacteria so I can't do Western Blots.

Does anyone know how I can confirm this is a CPS or if not what else it may be?

Thanks for reading this and any advice is much appreciated!!

Susan

Hey Guys,

Hopefully someone may be able to help me.

I am visualising my bacterial cells with a scanning electron microscope (SEM) and a transmission electron microscope (TEM). I am not carrying out the imaging myself (as my lab does not have an EM ) but I need to fix the samples prior to transport.

I will be fixing the cells in 3% gluteraldehyde buffer (equal volumes of buffer to cell suspension), however I am unsure what concentration of cells are suitable for TEM, SEM. 0.5 McFarlands, 1McFarland....4McFarlands????

If the sample is too turbid I doubt I will see individual cells....however if it's too dilute we will see no cells!

Does anybody know if there's a guideline to cell concentration prior to SEM and TEM??

Thanks in advance for your help!

Susan

Hey guys,

I'm hoping someone may be able to help me.

My lab is a microbiology lab and there is no one here with Biochem experience, so I'd really appreciate it if someone could read this and let me know if I'm going in the right direction.

I am trying to quantify the urease activity of my bacterial cells using the Berthelot method and I want to express urease activity as uM urea hydrolysed / min-1/ mg-1 protein.

-Produced a standard curve ranging from 0-500uM of Ammonia.
-Pellet of bacterial cells contains 0.6mg of protein - cells are lysed in 200ul of lysis buffer and 1,000ul of Reagent A is added to the sample and incubated for 30mins;
-a 90ul aliquot of the sample is removed and mixed with 1,050ul of Reagent B; Absorbance measured.
Urease activity expressed as uM of urea hydrolysed/min^-1/mg^-1 of protein
0.6mg protein in pellet
0.6mg of protein in 200ul
0.6mg of protein in 1,200ul
0.045mg of protein in 90ul aliquot
If OD₅₇₀ is 0.03, extrapolation from standard curve; 60uM ammonia; representative of the 90ul sample.
As urease activity is expressed as uM of urea hydrolysed/min^-1/mg^-1 of protein
Total of 30mins; 60uM/30min= 2uM of urea hydrolysed/min/ 0.045mg of protein
1mg of protein= 1000ug protein. I had 0.045mg protein in my 90ul aliquot.
1000/0.045=22.22 therefore 44.44uM of urea hydrolysed/min/mg of protein.

Does this make sence?

Thank you for reading this and I'd appreciate any comments.
Susan