Clarifications re: pOpen_v4 RNase Inhibitor

Open DNA Collections
1

Hi all!

I’m part of a group that’s working on expressing several proteins from the Research in Diagnostics Toolkit and we have a few questions regarding the RNase Inhibitor constructs in the current distribution (MDT-v1_1), mostly the one in pOpen_v4 (i.e., ODC_0221 / BBF10K_000014)

  • We couldn’t find the T7 promoter (as specified here, I think), but we found a Tac promoter (together with LacO). May we confirm that this is the promoter for the gene of interest?
  • We also found the sequence for Maltose-Binding Protein, upstream of a TEV Protease recognition site. Since the expressed protein is 6xHis-tagged, we are curious whether this tag was included to help with solubility?
  • We are similarly curious regarding the function of bases 4389-5842 within the annotation, ‘E coli optimized RNAse Inhibitor’

Lastly, we’d also like to know the differences, if any, between the two RNAse Inhibitor constructs (i.e., one in pOpen_build [ODC_0225], and the other in pOpen_v4 [ODC_0221]).

Your input would greatly help.

Cheers,
Camille

2

Hi Camille,

  1. In that construct, the gene of interest is under the Tac promoter.
  2. The MBP would help in solubility as well as for alternative purification using amylose conjugated resins.
  3. That is the codon optimized sequence of RNAse Inhibitor

You can find the sequences of the inserts of those constructs in the reclone website which leads to the github page (Open-DNA-Collections/Molecular Diagnostics Toolkit/Platemaps/MDT-v1_1.csv at main · Reclone-org/Open-DNA-Collections · GitHub), run then through a molecular biology software application like snapgene and it can help annotate widely used sequences. Alternatively, you can also do Nucleotide BLAST.

The version that was shipped to the PH hub last year was MDT-v1_2. Unfortunately, the github page was still not updated with that but it contains the same insert but under a T7 promoter and inside the pTi vector.

If you need more technical support feel free to email me at aaron.macauyag@gmail.com.

Kind regards,

Aaron

2 Likes