/sci/ - Science & Math » Thread #11956285

208KiB, 1376x1264, peGFP.jpg

View Same Google iqdb SauceNAO

Anonymous Fri 31 Jul 10:35:48 2020 No.11956285 View Reply Original Report

Quoted By: >>11956328

How do I figure out how to design a plasmid in snapgene that has
>Ampicilin Resistance with constant transcription
>Ori for bacterial amplification
>A promoter that's only active once the transcription of a pathway for example IL-6
>And a Marker like eGFP for transcription activation
Like I understand the very basics, but I do just cobble together those sequences from other plasmids in the database?
And is there anything I'm missing that every plasmid just NEEDS to have to function?
I see stuff like Kozak sequences or CMV promoters in the database, but I can't make sense of those things yet

Anonymous

Anonymous Fri 31 Jul 2020 11:01:31 No.11956328 Report

Quoted By: >>11956336

>>11956285
I'm in the protein expression and purification side of things, sounds like it's a bit different from what you'd be doing if you're using GFP as a marker. More on the cell biology side. In any case, you're on the right path. Yup, you find a plasmid, cobble together sequences from other sequences in the plasmids in the database.
>And is there anything I'm missing that every plasmid just NEEDS to have to function?
I see stuff like Kozak sequences or CMV promoters in the database, but I can't make sense of those things yet
This is key. What you need to get whatever gene that encodes for your protein-of-interest under the control of a promoter. For structural biology and protein biophysics where you need tons of protein, you add IPTG, a non-hydrolyzable analog of lactose, which induces overexpression of T7 RNA poylmerase via dissociation of the lac repressor. Upstream of gene encoding your protein-of-interest is the T7 promoter site and voila, your protein-of-interest is expressed. Now you need to optimize expression conditions, but that's a different talk for a different day.

Anonymous

Anonymous Fri 31 Jul 2020 11:05:26 No.11956336 Report

Quoted By: >>11957235

>>11956328
>For structural biology and protein biophysics where you need tons of protein, you add IPTG, a non-hydrolyzable analog of lactose, which induces overexpression of T7 RNA poylmerase via dissociation of the lac repressor. Upstream of gene encoding your protein-of-interest is the T7 promoter site and voila, your protein-of-interest is expressed.
While this is very helpful actually, and I'll keep this in mind, a setup like this would lead to constitutive overexpression of my protein.
But I currently want to design a reporter plasmid that only transcribes my marker once transcription of my pathway of interest is activated.
>What you need to get whatever gene that encodes for your protein-of-interest under the control of a promoter.
So basically figure out which promotor leads to transcription of IL-6 and attach my GFP or marker of choice to that promoter?

Anonymous

Anonymous Fri 31 Jul 2020 17:47:13 No.11957235 Report

Quoted By:

>>11956336
>While this is very helpful actually, and I'll keep this in mind, a setup like this would lead to constitutive overexpression of my protein.
Absolutely.
>So basically figure out which promotor leads to transcription of IL-6 and attach my GFP or marker of choice to that promoter?
Yes, I think this would work.

Capcode	All Only User Posts Only Moderator Posts Only Admin Posts Only Developer Posts
Show Posts	All Only With Images Only Without Images
Deleted Posts	All Only Deleted Posts Only Non-Deleted Posts
Ghost Posts	All Only Ghost Posts Only Non-Ghost Posts
Post Type	All Only Sticky Threads Only Opening Posts Only Reply Posts
Results	All Grouped By Threads
Order	Latest Posts First Oldest Posts First

Your latest searches