Entries by Alan Huett (8)


New paper out! LRSAM1, the E3 ligase crucial for anti-bacterial autophagy.

After what seems like forever our new paper came out in Cell Host and Microbe. It's open access, so go and download it!

In this paper we show that LRSAM1 is an E3 ubiquitin ligase that recognises intracellular bacteria and initiates the autophagy cascade. The majority of anti-Salmonella autophagy is dependant upon ubiquitination of bacteria, but the E3 ligase responsible was unknown (E3s select targets for ubiqutination and therefore generate the specificity of the ubiquitination process). LRSAM1 generates this specificity via a LRR (Leucine-Rich Repeat) domain which binds to intracellular bacteria, and a RING domain which drives ubiquitination. Once ubiquitin is attached to the bacteria, the autophagy machinery is recruited by ubiquitin-binding adaptor proteins.

Interestingly, LRSAM1 also directly binds one such ubiquitin-binding adaptor protein, suggesting that it might have a dual role, both generating and amplifying the autophagy-promoting signal by adaptor recruitment.

I am sure there is much more to discover about the role of LRSAM1, including what bacterial molecules trigger LRSAM1 recognition and binding and the targets of ubiquitination. These were questions we tried to address, but were unable to obtain satisfactory experimental data to definitively answer. I look forward to reading more about LRSAM1 in the future.


Gel doc image using macro and averaging

The article on building a cheap gel-doc has been updated with links to the relevant macros and I added some tips and slight improvements we have made in-use.

Below is an example image captured from a real gel - the first PCR in the Huett Lab! No contrast enhancement or cropping obviously, but it looks pretty good to me! 

SYBRSafe stained 1kb marker and PCR products


Welcome, Adam Collins

Latest member of the Huett Lab, Adam Collins joins to begin his PhD. Adam will be studying effector proteins from Adherent Invasive E. coli (AIEC), a Crohn disease-associated pathogen.

This is an MRC-funded studentship in collaboration with Dr Rob Delahay (UoN). I am very excited to have Adam join the lab and looking forward to seeing some great stuff in the next 3 years.


Cheap, LED gel-doc using a webcam - updated

A gel-doc is an essential item in any molecular biology laboratory, however a ready-made gel doc can cost a lot and will likely use UV lamps.  UV lamps are bigger and heavier than available LED technology and also may induce damage to your DNA samples, especially when using long exposures for band cutting.  Cheap blue LEDs are now widely available emitting in the 450-490 nm range and arrays of small LEDs are bright enough for gel imaging.  The blue light emitted is safe to view and does not risk DNA photodamage.

I bought a new Syngene Ultraslim LED transilluminator which has two 54 diode arrays of 470nm LEDs - this illuminates the central area very evenly.  Whilst not super-cheap, at £300 it still beats a UV transilluminator by some margin.  If you wanted to go really cheap you could get a blue LED grow light (check hydroponic suppliers) or do a full DIY installation of LEDs from an electronics supplier.

SYBR-Safe stained gel image taken with cellphone camera - note reflection due to high ambient light and original shiny base surface. Gel courtesy of Dr Bill Wickstead.The LEDs easily illuminate gels stained with SYBR-Safe or similar dyes - see the photo taken with my phone camera.  The dark orange lid filters out all the blue light, giving a really "constrasty" picture.  You can partially replicate this effect with LEE 12 (yellow) or 15 (dark yellow) filters, but you might have to stack them to get the best effect.

The built-in filter allows you to use any unfiltered camera for image capture and so we purchased a cheap webcam and fitted it into an insulated shipping box.  The white interior of the box gave us problems with stray light, leading to strong reflections of the camera on the filter glass.  Also the base of the trans was a sheet of clear acrylic over black, the shiny acrylic was also too reflective.  Gel doc showing black lining and webcam, the orange filter lid is raised.

 Both of these problems were solved by the application of matte black surfaces - black plastic card to the base of the trans (cheap, waterproof and replaceable), and matte self-adhesive vinyl to the box interior (Avery vinyl is used for sign-making and is widely available on e-Bay or at model/craft shops).  The resultant enclosure is very black and reflections are absent.

Since the webcam is a tiny, cheap sensor images are quite noisy.  However it is possible to collect images at 10 fps or higher, therefore we collect 20+ images and average them together to generate low-noise final images. This is currently accomplished using ImageJ, Virtual-Dub and the ImageJ Video capture macro.

Gel images from the final setup to follow.


MRC-funded PhD studentship available

I am pleased to announce that there is an MRC-funded PhD studentship available in the Huett lab, starting in the 2012/13 academic year.  

The project will examine the role of novel effectors in host-pathogen interactions, with a strong initial focus upon cellular imaging.  The second supervisor will be Dr Rob Delahay, and a successful candidate can expect broad exposure to cutting edge research and technologies within both the School of Biology and Centre for Biomolecular Sciences.

This studentship is open to all qualified UK students or EU students who have been resident in the UK for at least the last 3 years (EU students who have not been in the UK for 3 years will only receive fees and no stipend).  Funding is for three and a half years.

Applicants should have a strong academic and practical science record, with an upper second-class (2i) Honours degree or better.  High-scoring MSc or MRes qualifications and/or previous research experience would be an advantage.

Detailed application information is availble at the University of Nottingham, School of Biology website, or enquiries can be directed to me using the Contact page on this site.