OpenInsulin - Revision history

Rachel: /* Partners - You? */

2023-06-27T13:30:47Z

Partners - You?

Rachel: /* Further reading and recommendations */

2023-06-27T13:30:01Z

Rachel at 15:02, 22 June 2023

2023-06-22T15:02:31Z

Rachel: /* Next plans */

2021-04-07T11:07:12Z

Next plans

Rachel: /* Next plans */

2021-04-07T11:06:43Z

Next plans

Rachel: /* What are the specific challenges of the project and has anyone done this before? */

2021-03-03T08:01:59Z

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

Rachel at 07:59, 3 March 2021

2021-03-03T07:59:58Z

Rachel at 07:59, 3 March 2021

2021-03-03T07:59:27Z

Rachel: /* Photos */

2021-02-16T21:54:54Z

Photos

Rachel at 21:54, 16 February 2021

2021-02-16T21:54:10Z

@@ Line 110: / Line 110: @@
 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
 Maybe '''YOU''' want to join in, too!?

@@ Line 121: / Line 121: @@
 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>
-[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>
+[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)
 [[Category:Work In Progress]]
 [[Category:Projects]]

← Older revision		Revision as of 15:02, 22 June 2023
Line 74:		Line 74:

	(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. 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.)
		+
		+
		+	'''<big>Latest news</big>'''
		+
		+	Both plasmids were brought to colleagues at the Counter Culture Labs in Oakland, California, when RA went there to do the micronucleus workshop (June 2023).
		+
		+	=)

	== Photos ==		== Photos ==

@@ Line 71: / Line 71: @@
 *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 [https://www.abcam.com/Glucose-Uptake-Assay-Kit-Colorimetric-ab136955.html
+*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.)

@@ Line 71: / Line 71: @@
 *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.
+*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
 (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.)

@@ Line 47: / Line 47: @@
 *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<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.

@@ Line 2: / Line 2: @@
 <br>
 At Hackuarium, the first actual results are just beginning:<br>
-We are happy to have obtained '''sequence confirmation for two OpenInsulin plasmids''' for bacterial expression of Winsulin and Proinsulin 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>
 Here are some more details about the single-chain product expressed from the synthetic construct

@@ Line 89: / Line 89: @@
 File:Pwin.png | Winsulin construct
 File:Plates4strainestabDMF UNIL.jpeg| Plates for new strains to UNIL DMF -80 freezer
-File:8628167025315710071702398139356625312940032n.png| The Open Insulin Project logo
+File:86281670_2531571007170239_8139356625312940032_n.png| The Open Insulin Project logo

← Older revision		Revision as of 21:54, 16 February 2021
Line 89:		Line 89:
	File:Pwin.png \| Winsulin construct		File:Pwin.png \| Winsulin construct
	File:Plates4strainestabDMF UNIL.jpeg\| Plates for new strains to UNIL DMF -80 freezer		File:Plates4strainestabDMF UNIL.jpeg\| Plates for new strains to UNIL DMF -80 freezer
		+	File:8628167025315710071702398139356625312940032n.png\| The Open Insulin Project logo
		+