Difference between revisions of "OpenInsulin"

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(Further reading)
(Next plans)
 
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<br>
 
<br>
 
At Hackuarium, the first actual results are just beginning:<br>
 
At Hackuarium, the first actual results are just beginning:<br>
We are happy to have '''sequence confirmation of two OpenInsulin plasmids''' from Alex Kelly
+
We are happy to have obtained '''sequence confirmation for two OpenInsulin plasmids''' for bacterial expression of Winsulin and Proinsulin from Alex Kelly in Australia.
 
<br>
 
<br>
'''Winsulin''' and '''ProInsulin
+
Here are some more details about the single-chain product expressed from the synthetic construct
'''<br>
+
'''[http://2017.igem.org/Team:Sydney_Australia/Description Winsulin]'''. <br> ProInsulin encodes the insulin gene in its more usual conformation<br>
We hope to produce and test these constructs, in order to confirm their function and provide positive controls for other open insulin efforts.
+
We hope to produce and test the Winsulin and ProInsulin encoded by these constructs, in order to confirm their function and provide positive controls for other open insulin efforts.
 
<br>
 
<br>
  
Line 15: Line 15:
 
==What is this all about?==
 
==What is this all about?==
 
Insulin regulates sugar use in the body, and diabetics have problems around insulin production or responses.<br>
 
Insulin regulates sugar use in the body, and diabetics have problems around insulin production or responses.<br>
Complications of diabetes run the gamut from swelling to death... etc...
+
It's a huge and growing problem, because of our consumer society. Costs for insulin treatments are a big problem, particularly in the USA. <br>
 
+
Complications of diabetes run the gamut from swelling to death... (this page is still under construction, also see links at bottom)
 
+
  
 
==Our goals==
 
==Our goals==
Line 24: Line 23:
 
# Demystify and explain the best practices toward any open science project
 
# Demystify and explain the best practices toward any open science project
 
# Produce Winsulin and ProInsulin for open access positive controls
 
# Produce Winsulin and ProInsulin for open access positive controls
 +
# Provide strains to express both these products for further more 'DIY' efforts in community laboratories.
  
==Why participatory research?==
+
==Why open participatory research?==
One means to confront anti-science sentiments is to pull more people in <br> learning through hands on experiences is crucial...
+
One means to confront anti-science sentiments is to pull more people in. <br> learning through hands on experiences (and experiments) is crucial, for impacts on future behaviour and beliefs... <br>
 +
at least that's what we think - it's all an experiment, after all. <br>
 +
<br>
 +
 
 +
Other concerns about risks of DIY efforts going off the rails are improved by DIT (Do It Together) strategies. <br> Transdisciplinary teams mean also that philosophical and ethical points can be discussed at the same time as the most relevant controls are being chosen.
  
 
==Why Open Data?==
 
==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.
+
By making all the data generated by the project open, we want to explore and promote an alternative to proprietary initiatives. <br> Open data supports literacy and fosters innovation by everyone, from citizens to scholars. <br> Open designs can still be produced for commercial ventures, and can make profits if there is a market for the product.<br> An open philosophy prevents waste. If 90% of startups fail, where do their R&D results end up?  you may well ask.
  
 
==What are the specific challenges of the project and has anyone done this before?==
 
==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.<br>
+
Essentially, the production of insulin should not be subject to [https://www.t1international.com/blog/2019/01/20/why-insulin-so-expensive/ patent], and is straightforward.<br>
 
However, quality control is the main challenge, as injection or ingestion at least, is essential for efficacy.<br>
 
However, quality control is the main challenge, as injection or ingestion at least, is essential for efficacy.<br>
 
When purification strategies are tested, some measure of how well a given product works ideally would be determined relative to a known working solution.<br>
 
When purification strategies are tested, some measure of how well a given product works ideally would be determined relative to a known working solution.<br>
Line 43: Line 47:
 
*Remy visit during biohacker meet-up in old Hackuarium (Anthony participated virtually from Berkeley)
 
*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.
 
*many discussion with International group, also via Message group and Mattermost.
*Alex Kelly to Geneva<br>
+
*Alex Kelly to Geneva for diabetes meeting in around November 2019.<br>
Plasmids finally to Hackuarium, during pandemic <br>
+
Plasmids finally to Hackuarium, during the first six months of the pandemic. <br>
 
*finally transformed into DH5alphas in around August 2020, for Winsulin and ProInsulin, both constructs based on periplasmic expression vectors.
 
*finally transformed into DH5alphas in around August 2020, for Winsulin and ProInsulin, both constructs based on periplasmic expression vectors.
*
 
<br>
 
  
 
===Current status===
 
===Current status===
  
Clones grown in DH5alphas and finally sequence confirmation has come through.<br>
+
Plasmid clones have been grown in DH5alphas and finally sequence confirmation has come through.<br>
 +
The design of the constructs means the Winsulin and ProInsulin products are fused to the 6-His-tag and will thus bind Nickel in columns to be purified. <br>
 +
They also include a fusion - the Ecotin signal peptide, in order to secrete the products to the periplasmic space.<br>
  
 
Complications from low copy number vector and GC-rich stretches overcome via midipreps and 'power read upgrades' for the sequencing.<br>
 
Complications from low copy number vector and GC-rich stretches overcome via midipreps and 'power read upgrades' for the sequencing.<br>
Looks great...<br>
+
Looks great... ;)<br>
gels with EcoRI/SphI and HindIII diagnostic results.<br>
+
gels with EcoRI/SphI and HindIII diagnostic results. (see below in photo gallery)<br>
 
and mini vs midipreps<br>
 
and mini vs midipreps<br>
  
sequences, for instance see these [http://wiki.hackuarium.ch/images/e/e7/Winsulin_midi_T7term.pdf winsulin traces], are very clean for almost 1200 clipped length (for this winsulin power read result, Eurofins Genomics for sequencing with T7 term)<br>
+
The obtained sequences, for instance, see these [http://wiki.hackuarium.ch/images/e/e7/Winsulin_midi_T7term.pdf winsulin traces], are very clean for almost 1200 bases (clipped length, for this Winsulin power read result, Eurofins Genomics for sequencing with T7 term)<br>
  
 
===Next plans===
 
===Next plans===
  
 
*plasmids to be put in BL21 (production ''E. coli'' strain)
 
*plasmids to be put in BL21 (production ''E. coli'' strain)
*induced with IPTG to express the protein
+
*the two new strains in this context will be 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.  
+
*bacterial 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
+
*The soluble proteins will be applied to a 5ml column containing a Ni-NTA resin. The resin will be washed extensively before 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.
+
*Winsulin and ProInsulin His-tags will be proteolytically cleaved using Tobacco Etch Virus (TEV) protease. That cleavage will remove the His-tag from the peptide.
 +
*Isolated products will be tested for activity. One possibility if we can access mammalian tissue culture facilities, would be to use a [https://www.abcam.com/Glucose-Uptake-Assay-Kit-Colorimetric-ab136955.html glucose intake assay]. (but this kit is costly and the cells would generally need fetal bovine serum, so a DIY alternative is worth investigating...)
  
 
+
(Oscar Vadas of unige for 'pro' production, with LIMNA lab discussion for tests in TC and mice. Unfortunately, UNIL lab with whom we made an agreement a year ago is soon closing down due to the retirement of the PI, so we still need to figure out best means to test product.  The academic connection was key to the initial proposal, in order to confirm function of these plasmids' products, but further discussions are still necessary.  Winsulin initial ELISA results are still of interest to obtain. <br> Then if all goes well, this initial production run will provide not only the BL21 producing strain, but positive control products for future development and tests in community lab settings.)
(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. <br> Then if all goes well, this initial production run will provide positive controls for future development and tests in community lab settings.)
+
  
 
== Photos ==
 
== Photos ==
 
<gallery mode="traditional">
 
<gallery mode="traditional">
  
 +
 +
File:IntlOpenInsulin17nov19.png| Counter Culture Labs, Berkeley, International OpenInsulin meeting (17nov19)
 +
File:IntlOpenInsulin3.png| Alex Kelly, Australia (17nov19 - might also find Geneva pic... to confirm ok!)
 +
File:OpeninsE15dec19.png| Alex Kelly, Australia (15dec19)
 
File:OpenInsulinfilterpapers withplasmidsfromAK.JPG| DNA spots on filter papers from Alex in Australia
 
File:OpenInsulinfilterpapers withplasmidsfromAK.JPG| DNA spots on filter papers from Alex in Australia
 
ForValerie.png| gel shown for Colombian talk on Open Participatory research
 
ForValerie.png| gel shown for Colombian talk on Open Participatory research
Line 80: Line 88:
 
File:Ppro.png | ProInsulin construct  
 
File:Ppro.png | ProInsulin construct  
 
File:Pwin.png | Winsulin construct
 
File:Pwin.png | Winsulin construct
 +
File:Plates4strainestabDMF UNIL.jpeg| Plates for new strains to UNIL DMF -80 freezer
 +
File:86281670_2531571007170239_8139356625312940032_n.png| The Open Insulin Project logo
 +
 +
  
  
Line 86: Line 98:
 
==Partners - You?==
 
==Partners - You?==
  
Great thanks to the OpenInsulin Foundation and OpenInsulin project members everywhere.
+
Great thanks to the OpenInsulin Foundation and OpenInsulin project members everywhere.<br>
 
+
Thanks also to colleagues at UNIL's DMF and the LIMNIA network of diabetes researchers.
This project was initiated in Hackuarium by
+
[[User:Rachel]]
+
  
 +
This project was initiated in Hackuarium by [[User:Rachel|  Rachel Aronoff]]<br>
 
Martin (from Ireland and JOGL) and Vadim (from Estonia and OpenInsulin International) have joined in the discussions in slack.  Here is a  [https://join.slack.com/t/hackuarium/shared_invite/zt-lwmx9irq-7W9n7MdAgGkXXPMwVgVZUw 30 day invite to the slack channel] hackuarium.slack.com #openinsulin  
 
Martin (from Ireland and JOGL) and Vadim (from Estonia and OpenInsulin International) have joined in the discussions in slack.  Here is a  [https://join.slack.com/t/hackuarium/shared_invite/zt-lwmx9irq-7W9n7MdAgGkXXPMwVgVZUw 30 day invite to the slack channel] hackuarium.slack.com #openinsulin  
  
 
Maybe '''YOU''' want to join in, too!?
 
Maybe '''YOU''' want to join in, too!?
 +
write us at hello (at) hackuarium (dot) ch
  
==Further reading==
+
==Further reading and recommendations==
links<br>
+
Google Anthony Di Franco, who is inspiring... <br>
Here is a [https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(18)30200-2 very good article] with many useful links.
+
Also the Open EpiPen guy, Michael Laufer, (epipencil? fourthievesvinegar.org) has good things to say. <br>
Here is some [https://www.diabetes.org/blog/history-wonderful-thing-we-call-insulin history about Insulin].
+
Here is a [https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(18)30200-2 very good article] about insulin with many useful links.<br>
Here is the Wikipedia article about the OpenInsulin project
+
Here is some [https://www.diabetes.org/blog/history-wonderful-thing-we-call-insulin history about Insulin].<br>
 +
Here is the [https://en.wikipedia.org/wiki/Open_Insulin_Project Wikipedia article] about the OpenInsulin project. <br>
 
There was controversy after the Nobel, in particular about [https://academic.oup.com/clinchem/article/48/12/2270/5642437 giving the student credit also]!<br>
 
There was controversy after the Nobel, in particular about [https://academic.oup.com/clinchem/article/48/12/2270/5642437 giving the student credit also]!<br>
[https://www.diabetes.org.uk/about_us/news_landing_page/first-use-of-insulin-in-treatment-of-diabetes-88-years-ago-today#:~:text=Insulin%20was%20discovered%20by%20Sir,than%20a%20year%20or%20two. use of insulin] will be 100 years old in a year (2022)<br>
+
[https://www.diabetes.org.uk/about_us/news_landing_page/first-use-of-insulin-in-treatment-of-diabetes-88-years-ago-today#:~:text=Insulin%20was%20discovered%20by%20Sir,than%20a%20year%20or%20two. use of insulin] will be 100 years old in just a year (2022)<br>
  
  

Latest revision as of 11:07, 7 April 2021

The Open Insulin project is part of an international collaboration.
At Hackuarium, the first actual results are just beginning:
We are happy to have obtained sequence confirmation for two OpenInsulin plasmids for bacterial expression of Winsulin and Proinsulin from Alex Kelly in Australia.
Here are some more details about the single-chain product expressed from the synthetic construct Winsulin.
ProInsulin encodes the insulin gene in its more usual conformation
We hope to produce and test the Winsulin and ProInsulin encoded by 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.
It's a huge and growing problem, because of our consumer society. Costs for insulin treatments are a big problem, particularly in the USA.
Complications of diabetes run the gamut from swelling to death... (this page is still under construction, also see links at bottom)

Our goals

  1. Pave the way for more OpenInsulin tests with International Colleagues
  2. Demystify and explain the best practices toward any open science project
  3. Produce Winsulin and ProInsulin for open access positive controls
  4. Provide strains to express both these products for further more 'DIY' efforts in community laboratories.

Why open participatory research?

One means to confront anti-science sentiments is to pull more people in.
learning through hands on experiences (and experiments) is crucial, for impacts on future behaviour and beliefs...
at least that's what we think - it's all an experiment, after all.

Other concerns about risks of DIY efforts going off the rails are improved by DIT (Do It Together) strategies.
Transdisciplinary teams mean also that philosophical and ethical points can be discussed at the same time as the most relevant controls are being chosen.

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 everyone, from citizens to scholars.
Open designs can still be produced for commercial ventures, and can make profits if there is a market for the product.
An open philosophy prevents waste. If 90% of startups fail, where do their R&D results end up? you may well ask.

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

Essentially, the production of insulin 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 for diabetes meeting in around November 2019.

Plasmids finally to Hackuarium, during the first six months of the pandemic.

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

Current status

Plasmid clones have been grown in DH5alphas and finally sequence confirmation has come through.
The design of the constructs means the Winsulin and ProInsulin products are fused to the 6-His-tag and will thus bind Nickel in columns to be purified.
They also include a fusion - the Ecotin signal peptide, in order to secrete the products to the periplasmic space.

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

The obtained sequences, for instance, see these winsulin traces, are very clean for almost 1200 bases (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)
  • the two new strains in this context will be induced with IPTG to express the protein
  • bacterial 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 a Ni-NTA resin. The resin will be washed extensively before protein elution with a high-imidazole containing buffer
  • Winsulin and ProInsulin His-tags will be proteolytically cleaved using Tobacco Etch Virus (TEV) protease. That cleavage will remove the His-tag from the peptide.
  • Isolated products will be tested for activity. One possibility if we can access mammalian tissue culture facilities, would be to use a glucose intake assay. (but this kit is costly and the cells would generally need fetal bovine serum, so a DIY alternative is worth investigating...)

(Oscar Vadas of unige for 'pro' production, with LIMNA lab discussion for tests in TC and mice. Unfortunately, UNIL lab with whom we made an agreement a year ago is soon closing down due to the retirement of the PI, so we still need to figure out best means to test product. The academic connection was key to the initial proposal, in order to confirm function of these plasmids' products, but further discussions are still necessary. Winsulin initial ELISA results are still of interest to obtain.
Then if all goes well, this initial production run will provide not only the BL21 producing strain, but positive control products for future development and tests in community lab settings.)

Photos

Partners - You?

Great thanks to the OpenInsulin Foundation and OpenInsulin project members everywhere.
Thanks also to colleagues at UNIL's DMF and the LIMNIA network of diabetes researchers.

This project was initiated in Hackuarium by Rachel Aronoff
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!? write us at hello (at) hackuarium (dot) ch

Further reading and recommendations

Google Anthony Di Franco, who is inspiring...
Also the Open EpiPen guy, Michael Laufer, (epipencil? fourthievesvinegar.org) has good things to say.
Here is a very good article about insulin with many useful links.
Here is some history about Insulin.
Here is the Wikipedia article about the OpenInsulin project.
There was controversy after the Nobel, in particular about giving the student credit also!
use of insulin will be 100 years old in just a year (2022)