In general terms, the vector behavior of the vector is expected to be similar to the popular vector pET28a as they have the same plasmid replication machinery (pBR322 plus ROP gene) and the same lacI gene (with gtg start codon to improve the regulation without increasing the toxicity). In other words, the plasmid copy number, the amount of lacI repressor, and the T7RNAP level relation with the copies of the gene to be overexpressed are as in the pET28a expression system.
We characterize LacI regulation of pTI vector by cloning sfGFP reporter protein under pLac promoter and observed optimal performance (wide dynamic range and minimal leaky expression). Moreover, we try out some combinations of collection elements to carry out the expression of sfGFP in BL21 strain, aiming to find a workhorse set of them to overexpress enzymes of interest with standard requirements and facilities. It is: AB_pT7_Lac0 + BN1_Riboj54_B0034_His_tag + N1C_GS_linker + CD_Enzyme_Coding_Sequence + DE_3xStop + EF_Tz terminator, assembled in pTI acceptor vector.
Characteristics:
Improved T7 promoter with a LacI box which boost its regulation
RiboJ insulator to reduce the RBS activity variation given by the different 5’UTR that surround it in each particular assembled sequence.
B0034 middle strength RBS
6x Histag to carry out the purification in Histrap columns
12 residues GS flexible linker
improved T7 RNAP transcriptional terminator (Tz)
With this arrangement we have carried out the over-expression and purification of sfGFP, BstLF and HIV-RT successfully
Unfortunately, I didn’t have a successful transformation with the two pTi plasmids that I currently have. I am planning to retry it with the material I have still left, but I don’t have the place to do that yet.
I will post my update here.
Little re-correction here: I think @jenny_molloy may have mistyped me (YKH) when tagging for @YHK!
(Not the first time this has happened xD )
Apologies for the delay in replying to you, Yann (I hope that’s correct?) - we’ve been juggling a lot of things for Reclone behind the scenes recently, but hopefully we can share more in the near future.
As Jenny says, let us know which enzymes you’re testing, and hopefully we can help a bit more.
@FernanFederici@Isaac_N and @fxbuson also have good experience with working with pTi - perhaps @YHK, you can give more details with what DNA sequences you’re working with, the strain you’re transforming the pTi plasmids into, and any other details that might be helpful, and they may be able to provide you with more support?
Please feel free to directly message me your contact details and we can make arrangements to send you fresh plasmids also.
yes, indeed i did a transformation to top10 and DH5a with pTi & pOBL and till now i only exressed with pOBL an enzyme with 90 KDa with 2300 bp length and it works just fine, i wil let you know about the pTi once i try it, but the strain that worked for me is BL21 Rosetta it gave better results then other strains. @jenny_molloy@YKH
Hi I am working on expression of the Open Enzymes set in E. coli. Would it be possible to get pTi plasmid to experiment with? I run a nonprofit lab in New York and we have a project going to be a source for the Open Enzymes collection and take it further, funded through Experiment.com. My email is ejorgensen@biotechwithoutborders.org.
We’d be happy to share the pTi plasmid with you - can you give us more details via our ordering form? (pTi isn’t currently on the list, so do note it in the “special request” section.)
Hi everyone, following up on what Isaac posted, we think it would be useful to let everyone know what we’re doing with the plasmid.
We’re trying to verify that it’s a good backbone for our protein expression systems, specifically trying to express proteins from the Research in Diagnostics Collection (RiDC, previously Molecular Diagnostics Toolkit). The version they’re in right now should be “ready-to-express”, but wasn’t expressing quite as much as we would like. We’ve seen some promising results by just exchanging the entire insert gene from some RiDC plasmids to pTi, so we’re currently working on exchanging backbones for the rest of the collection.
I’ve had problems trying to join your meetings. I’m always stuck in a waiting room and never get to join. It could be something I’m doing wrong. Do you have any suggestions?
I’m sorry that you’ve been having problems joining the meetings.
I believe there might have been a confusion in the meeting start times: our usual meeting times are at 14:00 UTC on the second Wednesday of the month. Though I realise we did change the time last month, and the date this month in order to accommodate for end of year/start of year madness!
We normally try to include the date/time using the Forum settings and an arewemeetingyet link when we email/post out the event, as these should give the appropriate time for your time zone. There is also an “Add this event to your Calendar” link on the https://reclone.org/meetings/ site, which should add the event to your Google Calendar (if you use that).
My apologies if we forgot to add the links/make appropriate changes this time.
We have plans to set up a Calendar on the Forum too, so hopefully that’ll reduce any further time mix-ups!
@YHK we did our first Golden Gate assembly last night with pTiR. Transforming today, will report on our efforts later this week. Used several parts from the E. coli Expression Toolkit kindly supplied by Scott Pownall. We are attempting to express T4 DNA ligase, DpnI, EcoRI and EcoRI methylase in this first round.
I’m excited to hear that we are working on the same genes. I’ve successfully cloned T4 ligase and DpnI. We’re seeing good expression of T4 ligase under the pTac promoter, although there’s significant background expression in pDEST, which we’re still trying to optimize. As for the enzyme activity, we’re facing some challenges and haven’t yet assessed DpnI.
I wanted to reach out to you with a question regarding your work. Could you share which promoter you’re using for methyltransferases? I’m considering using a weak promoter for the recombinant production of restriction enzymes but haven’t decided on the best option yet.
Additionally, I’m interested in obtaining a copy of the pTi plasmid. Could you advise on how I might acquire it? I hope we can share our efforts and learn from each other’s experiences.
Sorry I took so long to answer! Sure, where are you located in the world? Are you okay with DNA on a paper strip? That is the easiest way to mail a plasmid to someone.
Also, we went with the simplest promoter option- the T7/LacO part from the E.coli Expression Toolkit. I have very little experience in enzyme production aside from my postdoc where I spent 3 days in a cold room with old fashioned columns isolating T7 and T3 polymerases! I can see where a weaker promoter would make sense for a restriction endonuclease. I think the plan is to cotransform with the EcoR1 methylase too…
Hello and sorry for the very long reply to update
Unfortunately the pTi backbone with phusion polymerase and open vent genes didn’t give an expression.
We used different protocols and different strains for expression, we are still trying to work it out.
@Ahmed_Atef did you have any further luck with DpnI? We have some groups in Cambridge that want to make this but we don’t have it in an expression construct yet! Which methylation-deficient strain were you using for expression?
@joseph_shenekji sorry to hear that you haven’t had any luck with pTi and Pfu and OpenVent, we’ve had success with OpenVent following the plate-based expression protocol with autoinduction media.
I would have to defer to @fxbuson for what he saw with Pfu expression in pTi.
@jenny_molloy yes dr. Jenny
But the pOBL backbone gave good results especially the open vent, i suspect my expression strains are old and can’t handle big plsmaids over 3.5 kb, i will try again with a new strains and autoinduction media and share also what happens.
For DpnI, you need to use a strain without dam or dcm methylases, NEB has one strain (C2925), but that is more like a cloning strain. I succeeded in getting DpnI expression using this strain (albeit in smaller quantities I would normally get by using BL21). A better strain, which is JM110, is available around (https://www.addgene.org/49763/) that is more suitable for protein expression.
After this, you either can use a DE3 lysogenization kit to integrate DE3 module (which has T7 pol under Lac promoter control) so that you can utilize T7-based systems, or use a E. coli promoter. What I did was to clone DpnI after the tac promoter (a chimera of trp and lac promoters) to do expression.
I apologize for the delayed reply. I’ve been dealing with some medical issues since my last message, and I’ve also settled in Egypt during this period.
I’m still very much interested in sharing the pTi plasmid, and I believe we could collaborate and exchange our results. Over the next few weeks, I plan to resume cloning methyltransferase enzymes using a different Anderson promoter (from iGEM) in the pOpen vector. Hopefully, I’ll achieve some promising results this time.
Best wishes for both of us:)
Atef
