26

The image was not in the DNA as such, only as an abstract representation that could be converted into an image from knowledge of the code. Briefly, they encoded the image into DNA, using a couple of different strategies in which DNA represented pixels -- either with a single DNA base representing a pixel, or with a triplet representing a pixel. Knowing the ...


17

Just to add what might have been missing in the beautiful answer by @iayork. I just want to give a more simple picture of the encoding done in the E. coli DNA. First for the rigid strategy in which 4 pixel colors were each specified by a different base, suppose we have a sequence: AAGCCCTGGTCAGCT Ignore the first AAG and start with C. Now, each base of ...


13

Purely going off experience here having used golden gate assembly methods for 5+ years now, there is a definite lack of literature regarding small part assemblies. In my current lab, we use CIDAR MoClo (a golden gate method) and have assembled 100's of constructs successfully with parts as small as ~ 70 bp. However, when we assemble smaller parts we tend to ...


11

That’s a great question and has lot of opportunities to explore. I am not sure anyone has followed up on this original BCD work systematically. We did try cloning these elements on a medium copy plasmids, in an operon design, driving multiple genes, and now I think about it, that was not a smart thing to do. Cloning was very challenging, probably due to ...


10

This image from the Kahn Academy article 'Overview of transcription' might help: Essentially, the sense/coding strand of the DNA encodes the sequence that is transcribed. The RNA polymerase binds to the antisense/template strand, for which the code is indeed TAC, but when it then transcribes this strand it is again complemented, giving us the AUG that is ...


9

Readthrough has already been used to implement transcriptional NOR gates, wherein tandem input promoters express a repressor that represses an output promoter. (First time I recall here: 21150903, used widely here: 27034378, roadblocking modeled here: 32141239.) An advantage of this design is that the repressor need only be encoded in DNA once, which can ...


9

I like this question, and I had a similar thought when reading that "Forward or Reverse Strand" post. I've since found a 2005 publication by Mirkin and Mirkin1, which investigates the interaction between the replication fork and transcription machinery in context of an E. coli plasmid. An excerpt from the abstract: Studying Escherichia coli ...


8

There isn't a simple answer. There are a lot of factors which can influence how stable a plasmid will be. I'll describe what the main factors are, but to work out exactly how stable your plasmid is, unfortunately you'll just have to test it. I've given links to some studies which do this at the end. Copy number Copy number refers to how many copies of your ...


8

I am René Inckemann and I am PhD student at the Max Planck Institute in Marburg, Germany. In my PhD I am using luminescence on a regular basis and I was also responsible to organize some of our Platereaders. So therefore I can tell you that you need a Platereader which is able to measure luminescence and you can not simply take a fluorescence platereader. ...


8

As the other answers here say, technically plate readers which are capable of fluorescent measurements can also make luminescent readings, but the sensitivity may be low (https://www.biotek.com/resources/technical-notes/use-of-the-fluorescence-optics-to-measure-luminescent-reactions-in-high-well-density-microplates-measuring-luminescence-in-1536-well-...


8

Viral methods are just more efficient and give you a higher viability in the end. Electroporation works but you'll have a low yield of viable cells after. Here's a nice paper looking at the effects of buffer composition on cell viability/transfection efficiency: https://www.nature.com/articles/s41598-020-59790-x with the general take away being, if ...


8

I don't have hard data to share regarding you question, but I can provide some anecdotal evidence. I have used golden gate assembly to build hundreds of plasmids using ~20bp annealed oligos, as well as hundreds of plasmids using ~70bp promoter+RBS, 720 bp eGFP, and ~70 bp terminators encoded on plasmids into a ~6kb backbone. In these cases I use 40 fmol ...


8

The PURExpress system from NEB is incredibly expensive, for this reason I have avoided it all costs during my experiments with cell-free systems. This is the paper I've used to create my own cheaper cell-free systems while also discussing reasons why using the NEB PURExpress system is not recommended: https://pubs.acs.org/doi/10.1021/acssynbio.8b00427 "...


7

If there is only one CDS, presumably there will be a transcriptional termination directly after the CDS (after the stop codon actually), which makes any read-through coming from using a single stop codon almost negligible.


7

I am currently working with these tags, so going off experience here. The binding efficiency is not 100% even when the correct versions are mixed e.g. version 2 spytag and version 2 spycatcher. I believe this is the original paper (https://pubs.acs.org/doi/pdf/10.1021/ja910795a) it reported 60 % binding efficiency after an hour (I presume for version 1) and ...


7

I have worked with the coupling of SpyCatchers and SpyTags of different iterations and can confirm that they are all backward compatible. While SpyCatcher003 and SpyTag003 pairs have the quickest reaction rate, reactions of all pairs (including SpyCatcher and SpyTag version 1) should go to completion under the right conditions. The SpyCatcher002-SpyTag1 is ...


6

There's no rule that says a transcription factor must be either a repressor or an activator. The lambda repressor (CI) is in fact a repressor and activator of transcription, depending on where it is bound and to what promoter you are referring to. I know your question isn't directly about lambda phage, but I think this mechanism may be best explained in the ...


6

Since a few people asked why the AAG triplet is avoided in the code, I thought I'd add this in addition to the other answers. The interesting part of this research is not necessarily the image encoding but rather how they utilized the CRISPR system to integrate the encoding DNA into the genome. It may be a surprise to some that the image is not encoded in ...


6

The impact of any read-through from a leaky stop codon in an expression unit with only one CDS would probably depend on a few things, mainly (i) where is the next in-frame stop codon, (ii) what are you trying to express, and (iii) how leaky is the stop codon? In cases where the next in-frame stop codon is only a few base pairs away, there would probably be ...


6

Perhaps I could add for eukaryotic systems which terminate all stop codons by a single protein, eRF1, this study by Schmied et al., showed readthrough of all stop codons to be less than 0.2% by wild-type eRF1, which seems acceptably low! I am unsure how this would transfer to prokaryotic systems which diverges in its use of two release factors - RF1 and RF2. ...


6

From a technical perspective, for the FlopR package you mention (and I believe also for Jacob Beal's protocol) you can simply change the bead size and perform a new calibration. The only information that you need is the bead count at each point of your calibrant curve. However, using calibration beads of a given size assumes that your cells will remain ...


6

Some clarifications: Commonly used models for bacterial isothermal growth curves (which represent the growth curve directly) are for example: Exponential Logistic Generalized logistic The Monod equation expresses how the growth rate changes with the availability of substrate see a related question here. The Monod equation can be incorporated into an ...


5

I'm tempted to say, "It's complicated." CI does indeed act as both a repressor and activator. Transcription regulation in the lambda bacteriophage is quite complex for such a small system, so some confusion is understandable. Lewis et al. gives a rough description in a relatively recent paper (1): The CI protein autoregulates its synthesis. At low ...


5

I'm afraid there's not much of answer but back to the bench with you! (That's why it's called re-search). There are several factors that could be playing a role here. You could have transcriptional or translational read through. You should check your sequence and the cells you are going into to make sure you aren't getting Amber (UAG) suppression. ...


5

By looking at the sequence only? This is an unsolved problem. Its not clear to me exactly what you are looking for, but here are some thoughts... On a relatively narrow question as to whether any gene you plug into a bacterium, yeast, mouse, goat or other transgenic organism - the rna may not translate into protein in detectable levels or at high enough ...


5

Obviously, one can't use antibiotic resistance since they are not bacteria. This is not so obvious. Some common antibiotics are active against archaea.1 The Halohandbook, an indispensable document for the propagation and transformation of Halobacteria, includes a list of selectable markers and their concomitant plasmids: NovR, MevR, and TmR are genes ...


5

Aside from the comment that frozen cells require less manual maintenance and expense (which is a big plus), active cultures can rearrange/recombine plasmids causing mutations and deletions (especially in the case of toxic inserts). For example, see this article: Peijnenburg AA, Bron S, Venema G. 1987. Structural plasmid instability in recombination- and ...


5

the more biologically grounded Monod growth Why is the Monod model more biologically grounded? It is empiric and NOT based on biological considerations! I only know this equation from enzyme kinetics, not for cell-growth, where the identical Michaelis–Menten equation assumes a random collusion model. So according to this model, your cells would grow more, ...


5

It may also be worth considering how you are amplifying and isolating your small part. Gel extraction kits sometimes have an optimum DNA size ranges that may prevent recovery. Or if your part is similar to your primer length you may see considerable contamination in your 'purified' product if say the size of a single RBS.


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