I’m interested in developing an “Open Cloning Kit” based in part on "Producing molecular biology reagents without purification” paper by Dr Molloy.
We will be starting with the enzyme and ligase and once the replication works redesign the enzyme as a one plasmid and incorporate some open devices in the protocol to offer a complete solution.
I have several people already interested in joining the project however of anybody else is interested I will be more than happy to include you.
This open kit could be used both for research and for education and I would expect the project to be developed in an iterative manner.
The paper is amazing and very broad in scope.
This will not include all the experiments in the paper but just the MoClo part.
You can reply here or email directly gmail to openbioscience/
Thank you for sharing this exciting initiative. I’m very interested in contributing to the Open Cloning Kit project.
I have access to a lab facility and some helping hands (my students) here in Egypt, and we can carry out experiments to support the development, particularly the MoClo part you mentioned. I’d be glad to collaborate and test the protocols in our setup.
Looking forward to following the progress and discussing how we can best get involved.
Thank you for sharing this opportunity. My group and I are interested in being part of the project. I am from INSIBIO-CONICET, Argentina.
Saludos cordiales,
PhD. Guillermo A. Vega Lopez
Universidad Nacional de Tucuman
Chacabuco 461, Tucuman
(CP: T4000ILI) -Argentina
Web of Science ResearcherID: M-3403-2014
ORCID: 0000-0002-2426-2844
Sounds interesting. Glad to collaborate in the project. Here in UPC (Lima, Peru), we work routinely in recombinant protein expression and purification. Additionally, we’re starting to work with open source devices.
We have an overwhelming interest in this project so far.
There is a great number of quality participants. I also have a number of people that are not yet in the Reclone sphere that are equally interested.
Several thoughts to start with:
This will probably deserve a meeting of its own so we’ll have to organize that.
It would be good to start a draft high level document.
As a project it should have an online place to collect materials, probably GitHub.
Also since this a project that might require instant communication please let me know if I should create a Slack group.
Keeping a project with so many participants running will require dividing the work based on capabilities, availability and interest.
Great initiative. Please count us in from Aysén, Chile.
We have the basic infrastructure to help build and test low-cost kits, and we have been working with OpenFlexure microscopy and other low-cost/open-source devices for education and research. This project would fit very well with what we are trying to develop here.
Dr. Jorge A. Toledo
Profesor Asociado
Departamento de Ciencias de la Salud
Dirección: Eusebio Lillo 667, Coyhaique. Chile
ORCID: ORCID
It’s been an overwhelming response in interest from more people and labs that I expected. Despite being seemingly quiet I’ve been working in the background to get more valuable participants from outside this circle and having several individual meets with people I thought worth attracting.
I’m waiting for a couple of replies that I hope will clarify shipping options.
Following recent feedback, I want to clarify and tighten our project scope. We are pivoting to develop this as an educational kit.
This shift narrows our focus by cutting out alternative avenues that would complicate testing. However, it increases the need for robust, highly structured documentation and protocol design. While this requires more upfront writing, it gives us a cleaner, more standardized path for testing.
Here is the updated, lean breakdown of what we need:
Biological Materials and Strains
Chassis: endA-, recA- E. coli BL21(DE3) strain. Not available now in my lab. Do any of you happen to have it?
Plasmids: pBR BsmAI-M Kan + p15A BsaI-R Amp (currently coordination-sourcing these from Dr. Jenny Molloy).
Test Chassis: A plasmid featuring two BsaI recognition sites downstream of a Lac promoter (we might use FreeGenes directly for this). Reporter: The simplest possible reporter (such as a chromoprotein or superfolded FP) under an open MTA, flanked by BsaI sites.
Downstream Processing and Reagents
No Lyophilization: We are ditching freeze-drying in favor of a straightforward air or heat drying technique.
No Chromatography: Dry reagents will be used directly. We will still include an affinity tag if available, but skipping purification slims down the protocol.
Cell Drying Setup: I have a low-contamination cell drying setup using very basic tools. Action item for me: I will send you photos and a draft protocol shortly.
Hardware and Lab Requirements (Minimalist Approach)
Culturing: An incubator and standard media. We want to see if we can get by with a simple tube rotator instead of an incubated orbital shaker to keep user requirements low.
OD Measurement: We will attempt to bypass cell density devices by relying purely on standardized incubator timing.
Induction: Starting with IPTG, with a plan to switch to lactose later for better cost and global availability.
The Big Picture:
The entry barrier for this kit should be incredibly low. Most places should easily meet these basic hardware and reagent thresholds.
What are your thoughts on this direction? I just finished my conference work, so I finally have the bandwidth to dive into the protocol draft.
I’ve been teaching biotech to the public in low resource settings for the past 25 years. I have one suggestion.
Relying on timing is okay for some things but temperature and other factors can play a big role in how well your competent cell prep or expression works, and knowing the OD is super useful (yes I have used rotators and guessed but we want this kit to be easy to get good results with).
In the old days we used to use something called a Klett to measure cell density. It was basically a light bulb in a box and an analog needle display. You will see Klett readings in papers from the 1970s and earlier. Something like that should be easy to DIY and might be helpful to at least establish timing of growth in someone’s particular setup.
You are right that timing is not very reliable for many cases. The “open kit” shall not require users to create competent cells but expression will be influenced by the incubation temp quantity and obviously the incubator shall control the temp reasonably well. We should have an OD operating range. The classic example @37C is in the 0.4 -0.6 and maybe timing (12 to 15 hours) will be enough to fit the range. Maybe even cultures passing the log phase will work as dry cells are ‘dirty’ systems to begin with.
Thank you for the Klett suggestion. I have not used it nor have I seen one and it might be an option for some users especially if it would be easy to procure or build
Personally I think the best and simplest option would be the glass standards, but they seem to be expensive.
The other option will be McFarland standards but it would require an additional reagent. Maybe the best option for some users.
In the past I have not been very successful in counting the small e-coli cells using a hemocytometer. That would also require a quality microscope and potentially some staining reagent most likely carcinogen. But it is a potential option too.
I have developed and used an inexpensive Optical Density meter https://github.com/AdrianMolecule/bioreactor/tree/master/OdMeter that is simple to use yet not totally free and it requires some building work.
Testing and optimization phase work shall deal with this.