OpenInsulin

The Open Insulin project is part of an international collaboration.
At Hackuarium, the first actual results are just beginning:
We are happy to have sequence confirmation of two OpenInsulin plasmids from Alex Kelly
Winsulin and ProInsulin
We hope to produce and test these constructs, in order to confirm their function and provide positive controls for other open insulin efforts.

The project has been under discussion for quite some time, and we hope more people will want to join in for this work.

What is this all about?

Insulin regulates sugar use in the body, and diabetics have problems around insulin production or responses.
Complications of diabetes run the gamut from swelling to death... etc...

Our goals

Pave the way for more OpenInsulin tests with International Colleagues
Demystify and explain the best practices toward any open science project
Produce Winsulin and ProInsulin for open access positive controls

Why participatory research?

One means to confront anti-science sentiments is to pull more people in
learning through hands on experiences is crucial...

Why Open Data?

By making all the data generated by the project open, we want to explore and promote an alternative to proprietary initiatives. Open data supports literacy and fosters innovation by both citizens and scholars. Open designs can still be produced for commercial ventures, and can make profits if there is a market for the product.

What are the specific challenges of the project and has anyone done this before?

Essentially, the production of such molecules should not be subject to patent, and is straightforward.
However, quality control is the main challenge, as injection or ingestion at least, is essential for efficacy.
When purification strategies are tested, some measure of how well a given product works ideally would be determined relative to a known working solution.
This is what we hope will be only a first contribution by Hackuarium towards the OpenInsulin collaboration.

Open Insulin history
Hackuarium's Open Insulin Entanglements

first got aware about this project and discussed potential studies during the biofabbing events
Remy visit during biohacker meet-up in old Hackuarium (Anthony participated virtually from Berkeley)
many discussion with International group, also via Message group and Mattermost.
Alex Kelly to Geneva

Plasmids finally to Hackuarium, during pandemic

finally transformed into DH5alphas in around August 2020, for Winsulin and ProInsulin, both constructs based on periplasmic expression vectors.

Current status

Clones grown in DH5alphas and finally sequence confirmation has come through.

Complications from low copy number vector and GC-rich stretches overcome via midipreps and 'power read upgrades' for the sequencing.
Looks great... ;)
gels with EcoRI/SphI and HindIII diagnostic results. (see below in photo gallery)
and mini vs midipreps

sequences, for instance see these winsulin traces, are very clean for almost 1200 clipped length (for this winsulin power read result, Eurofins Genomics for sequencing with T7 term)

Next plans

plasmids to be put in BL21 (production E. coli strain)
induced with IPTG to express the protein
Cells expressing Winsulin/ProInsulin will be harvested by centrifugation and lysed using a French Pressure cell. The lysate will be centrifuged at 30'000g for 35min to remove cell debris and insoluble proteins.
The soluble proteins will be applied to a 5ml column containing Ni-NTA resin. The resin will be washed extensively before the protein elution with a high-imidazole containing buffer
Winsulin purified using the His-tag will be proteolytically cleaved using Tobacco Etch Virus (TEV) protease. That cleavage will remove the His-tag from the peptide.

(Oscar Vadas of unige for 'pro' production, with LIMNA lab discussion for tests in TC and mice. This is the initial proposal, in order to confirm function of these plasmids' products.
Then if all goes well, this initial production run will provide positive controls for future development and tests in community lab settings.)

Photos

DNA spots on filter papers from Alex in Australia
gel shown for Colombian talk on Open Participatory research
gel with H3 digest and midi vs miniprep
GC rich seqs
ProInsulin construct
Winsulin construct

Partners - You?

Great thanks to the OpenInsulin Foundation and OpenInsulin project members everywhere.

This project was initiated in Hackuarium by User:Rachel

Martin (from Ireland and JOGL) and Vadim (from Estonia and OpenInsulin International) have joined in the discussions in slack. Here is a 30 day invite to the slack channel hackuarium.slack.com #openinsulin

Maybe YOU want to join in, too!?