Which method of gene amplification for toehold switches?

Question

My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience in wet lab work. We plan to order 3 genes containing an arabinose inducible promoter, a riboswitch, a leuciferase CDS and a terminator. These genes are each ~3kb in length.

We plan to order our genes from IDT, but we're not quite sure which strategy to deploy to amplify these genes before adding them to a cell free extract for characterisation.

The four methods we have narrowed it down to are as follows:

1. Order the genes in plasmids, linearise, PCR, ligate, add to cell free extract for characterisation.
2. Order genes in plasmids, transform straight into E. coli, culture, mini prep, add to cell free extract for characterisation.
3. Order genes in plasmids, linearise, PCR, ligate, transform into E. coli, culture, mini prep, add to cell free extract for characterisation.
4. Order genes as gBlocks (not in plasmids), amplify with PCR, ligate into plasmid backbone, transform, culture, mini prep, add to cell free extract for characterisation.

We'd ideally like to use the method which is most simple as we will not have a lot of time in the lab, and we don't have much experience.

Any advice on choosing between these strategies would be much appreciated!

Answer 1

Option 2 is your simplest and easiest. It should give you unlimited plasmid to work from. Note that ordering pre-made sequences of this size isn't particularly cheap and might take a few weeks to get to your destination.

It may be easier (quicker + cheaper) to find a plasmid with the luciferase CDS and terminator already built in. You could then amplify this with primers incorporating your promoter and riboswitch sequences and some restriction enzyme sites to generate a product that looks like this:

RE1-ara-riboswitch-luciferase-RE2

You can then clone this into a(nother) plasmid cut with RE1 and RE2.

Another option is that if you have a plasmid with a luciferase gene in it already and a multiple cloning site (MCS) you can order complementary primers (note single-stranded DNA, anything less than about 150 bases is fine) containing the araC and riboswitch sequences flanked by two restriction sites, you then anneal the two primers, digest off the ends and ligate. You just have to be careful with making sure that everything is in-frame. I don't know anything about riboswitches though so frame may not matter so much.

As you have limited time I suggest buying chemically competent cells for heat-shock transformation (if you don't have some already); make sure you don't get electro-competent ones, as these will only work with an electroporator. Also get a commercial mini-prep kit (e.g. Qiagen plasmid mini-prep). These are easy to use and reliable.

I think in option 1 you have something a bit odd going on - why would you digest your plasmid then re-ligate it, when it is already in the form that you are going to use?

I am not quite sure why in options 3 and 4 that you think that these are any different in terms of the end result to option 2.

Answer 2

I basically agree with Bob, but wanted to add a few more details.

Out of the options that you have available, option 4 is the most common way to do it. I wouldn't consider options 1 and 3. I don't think option 1 will really work to produce the amount of plasmid that you need, option 2 is a much easier way of amplifying it. As for option 3, you shouldn't need to amplify the plasmid before transforming it, on the IDT website they say:

All of our synthetic gene products are provided in an optimized cloning vector that is ready to be transformed into E. coli.

Between options 2 and 4, it comes down to time and cost. Unless you have a deal for free synthesis from IDT, the cost of option 2 is going to be around £1100 vs £330 for the gBLOCK (although another consideration here is that the maximum gBlock length listed on the website is 3kb). In terms of timing, the website says it will take 5-8 business days before shipping a gBlock vs 25 business days for the plasmid. Someone who is an experienced cloner would definitely be able to clone the gBlock into a vector faster than that (might take as little as 1-2 days), but if you're first-timers you might find that it takes you months to get it to work (the first time I cloned it took me a long time).

Another factor for both options 2 and 4, depending on how you do it, is that if you're using different toehold switches then you'll either have to order 3 plasmids/gBlocks with essentially all the same stuff apart from the toehold switch, or replace the toehold switch using something like an inverse PCR.

Assuming you are an iGEM team, another option, although maybe not the best if you have free DNA synthesis from IDT, is that the iGEM distribution kit will be distributed around the end of June. This kit should contain samples of the luciferase CDS which you can amplify using PCR with primers to assemble into a vector of your choice. I would only recommend this method if you're limited on synthesis capability/are having issues with amplifying your gBlocks. You also wouldn't have the promoter/terminator/toehold switch, so you'd need to include them.

To summarise:

• The best way (if you have the funds) is option 2
• A slightly cheaper way is to order the full plasmid for one toehold switch and then use PCR to replace the toehold switch for the two others
• The 'normal' way that I in my experience is the way most people do it is option 4, this is also the quickest if you know what you're doing
• The cheapest way to do it is using the iGEM distribution kit