Production of OpenVent cellular reagent- protocol

Hi everyone i would like to share with you a protocol on local manufaturing of OpenVent cellular reagent.The enzymes are expressed in E.coli using IPTG as inducer and the dried cells are used as cellular reagent. It would be great to have your comments so as to make this protocol more comprehensible and useable.
You can also find here a link to a couple of protocols all used or needed during enzyme manufacturing, together with the data record forms for each SOP. .
Do feel free to bring feedbacks unto the forum for improvement on the methods for better results and easy comphrehension.

Hi Nadine and everyone I have some questions that will hopefully improve the protocol :slight_smile:
Not sure if to put it here or on the google doc…
I was wondering what do you commonly use as a desiccant, and maybe if there are some other options? Maybe things that would comonnoly be in a lab or that can be bought cheaply at a market?
Also was wondering if anyone has compared this to freeze drying? In terms of activity/stability?
Also maybe adding trehalose, which is know to help stabilize dry enzymes?
Lastly Is there a protocol somewhere for how to use it later? i,e what buffer and conditions work well for this enzyme?

Hello Guy,
Thanks a lot for your input and sorry for the delayed response.

To dry the enzymes we currently use commercial silica gel beads (Silica gel, 2-5 mm, Merk CAS-No:7631-86-9) as a desiccant. As for other options, we believe that the lyophilization procedure will work well with any solid desiccant potentially found in the market and which could be used without specialized handling techniques such as silica or anhydrous calcium sulfate (drierite). However, this needs to be experimentally validated, considering the fact that some desiccants could contaminate the reagents with inhibitory compounds. We are still investigating other cheap and locally available alternatives to silica.

We don’t have a direct comparison between silica and freeze-dried samples. However, we know that one difference is that cells will die during the freeze-dried desiccation process (unless trehalose is added) while cells dried with desiccant are alive for months.

We’ve been able to show the stabilizing effects of Trehalose on cellular reagents enzymes in Master mixes formulations but we didn’t try adding the trehalose before drying down the reagents, which is definitely worth trying.

A protocol to access the functionality of produced cellular reagents enzymes exist (SOP011). Cellular reagents work well with 10X Thermopol reaction Buffer following PCR parameters and conditions suitable for the enzyme being expressed and prepared.