My reading

After 2022-06-24 (when I started recording)

June 2022

Assembly of a nucleus-like structure during viral replication in bacteria

(2022-06-25) An very interesting phage that basically creates a protein nucleus.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads

(2022-06-25) Fascinating example of going towards full automation with digital microfluidics. They built an electrowetting system that can automate MAGE. The other very interesting bit is the use of bioactivated magnetic beads, rather than centrifugation, for the concentration of cells. I bet that can be used elsewhere for automation.

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

(2022-06-26) Having 100 electroporation chambers are very interesting because you could do optimization of electroporation on a single chip - especially useful for new and difficult to engineer organisms. However, they fabbed their own chips... Sounds insane. Is it really though? Might have to contact the original authors to find out.

Construction of a synthetic Saccharomyces cerevisiae pan-genome neo-chromosome

(2022-06-30) Fascinating example of large scale chromosomal engineering. They basically took the genes from wild strains of S.cerevisiae and plopped em on a synthetic chromosome, and then showed that the chromosome actually increased the metabolism of the strains. More interestingly to me is that they proved that SCRAMBLE could actually increase growth rates adaptively. Interesting lesson.

Everything You Ever Wanted to Know About Saccharomyces cerevisiae Telomeres: Beginning to End

(2022-07-02) Holy shit, telomeres are wack. Basically every gene associated with telomeric ends is doing double duty around the cell. Near the middle of this long and detailed review I found I wasn't learning too much (or at least, useful hooks I can come back to) so I'll reserve the rest for when I'm working directly with telomeres. Still, very well written.

Nix: A Safe and Policy-Free System for Software Deployment

(2022-07-05) Learned a little about Nix from the original paper. Basically, the idea is that shared libraries are kinda hard to deal with. So nix organizes shared libraries under a hash of the implementation, therefore, you will never have broken packages. Simple idea, kinda reminds me of Unison lang. Few problems I see with this approach: 1. native build methods aren't too friendly, so patches will have to be made 2. for many packages you'll have to rebuild like, the entire world. Those aren't that big of a deal, but for much of what I do, Nix would add to the complexity. Go already does approximately the hash-for-package thing in go.sum, and the rest of my systems are often stable enough that I never have to worry about random C packages. Still, very cool idea - perhaps I'll have to try it out sometime.

Electrochemically Generated Acid and Its Containment to 100 Micron Reaction Areas for the Production of DNA Microarrays

(2022-07-22) Very cool paper on how CustomArray (acquired by Genscript) makes their oligo pool tech work. Unlike Twist or Agilent, they use silicon chip technology - a very, very, very scalable technology - to build DNA. Not only that, but their patent is expired. It works by locally producing acid using a voltage switch, which can then selectively do biological reactions. I wonder how hard it would be to reproduce...

Helix: An Electrochemical CMOS DNA Synthesizer

(2022-08-07) A modern paper on electrochemical DNA synthesis. Too bad they're commercializing the tech with Avery Digital. The most interesting bit I got from the main author is the Al is incompatible with DNA synthesis chemistry, so we're gonna have to use platinum if we want to reproduce with normal chips, meaning a post-processing sputter-coating step. Annoying. They can also fit an absurd quantity of microwells on the chip, could likely fit about 1mil on a normal size chip.

Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation

(2022-08-26) Another insane Japanese group figured out how to work with genomes in-vitro. Very cool work - basic idea is to use replication machinery to stabilize genomes while working with them in-vitro. This could be very useful for working with large DNA fragments.

A History of Erlang

(2022-12-16) A fascinating history of Erlang, a concurrent programming language. The overall theme I learned can be summed up with the quote: "An Erlang system can be thought of as a communicating network of black boxes. If two black boxes obey the principle of observational equivalence, then for all practical purposes they are equivalent. From this point of view, the language used inside the block box is totally irrelevant. ... If the language inside the black boxes is of secondary importance, then what is of primary importance? I suspect that the important factor is the interconnection paths between the black boxes and the protocols observed on the channels between the black boxes." This influences about how I think about large systems for biological protocols - because things can take so much time (biology!), concurrency management of when protocols start and stop is much more important, especially making sure that is reliable and understandable. I think Erlang gives a real good way to do this.

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics

(2022-12-18) Isothermal DNA amplification. In some ways, it is better than PCR - faster, more processive. I can't explain how big this could be: it replaces hardware with enzyme.

Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

(2022-12-18) You can PCR for circular DNA if you add in a ligase. Yea, makes complete sense. Bet you could amplify circular plasmids with it.

Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA ass…

(2022-12-18) 10 part DNA assembly without enzyme. Takes a ton of primers, but honestly the idea is kinda genius.

The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis

(2022-12-18) PIPE DNA assembly. Uses just PCR for DNA assembly. Super simple - bet could be easy to implement.

(Catching up on some old papers 2023-03-25)

A set of experimentally validated, mutually orthogonal primers for combinatorially specifying genetic components (2018)

(2023-03-25) Mutually orthogonal primer set for PCRing things out of a pool. We used this for FreeGenes! Basically, given n primers, you can PCR 0.5*n*(n-1) unique sequences. We also called this "dial-out" PCR. They experimentally validated their set, which makes them much more useful.

Producing molecular biology reagents without purification (2021)

(2023-03-25) This paper is validating "cellular reagents", which is basically just the method of using enzymes from highly expressing strains without a purification step - you just dessicate 'em! It's a very cool and promising method for low cost reagent production. In this one, they show both BsaI and T4 ligase cellular reagents are functional... O_o

Construction of a Super-Competent Bacillus subtilis 168 Using the PmtlA-comKS Inducible Cassette (2015)

(2023-03-25) They made an inducibly competent Bacillus subtilis in the presence of mannitol. Most interestingly, they deleted manPA for their gene cassette, which means that you can use mannose negative selection in their strains. So you have a Bacillus subtilis with two very interesting abilities: natural competence, and potential for negative selection! I believe this strain is used in the mini-bacillus project.

Large-scale DNA-based phenotypic recording and deep learning enable highly accurate sequence-function mapping (2020)

(2023-03-25) They linked RBSs to translation/transcription, then tested the strength of hundred-of-thousands of RBSs at once using sequencing. Generally, I think this is an extremely powerful method to measure things: measuring things with sequencing resolves to essentially single copies, and you can massively pool them in cells.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance (2016)

(2023-03-25) One of the most exciting uses of PACE ever. They evolved Bt toxin to overcome resistance using phages. Definitely one of my fav papers of all time, very creative!

Low-N protein engineering with data-efficient deep learning (2021)

(2023-03-25) A perfect demonstration of why AI is cool with biology. With only 24 intelligently assayed GFP, they were able to essentially make a sfGFP! They did this by training the model on all of protein space to screen out "unnatural" mutations... and it somehow fucking worked. Wild!

MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme (2018)

(2023-03-25) They used a single type IIS restriction enzyme to do hierarchical DNA assembly. I bet this can be combined with more aggressive yeast-toolkit type linkers to get a really cool system going...

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes (1986)

(2023-03-35) The OG BL21 paper.

In vivo hypermutation and continuous evolution (2022)

(2023-03-26) A good primer on in-vivo directed evolution / continuous evolution. I used to work in liulab, so I am biased towards thinking OrthoRep is cool, but MutaT7 also looks quite cool! It has a very easy implementation route, which I should check out.

Machine learning for optimization of multiscale biological circuits (2023)

(2023-03-26) A quite good paper going over modeling for biological circuits! I like the declarative approach a bit better, but this still makes sense. Basically they use MATH to simulate circuits more effectively.

Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production. (1984)

(2023-04-13) A chemically defined media for growing Bacillus subtilis + having them sporulate. Easier than it sounds - Bacillus needs certain chemical ratios in order to sporulate. Their broth is called CDSM, which I'm currently looking at making, so I can have all defined media for my workflows!

Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design (2022)

(2023-04-13) Scientists used GoldenGate + ligase fidelity calculations to assemble 52 parts at once. Important data for me - 52-fragment assembly reaction produced ~800-fold fewer plaques than the equivalent 10-fragment assembly reactions of the same target. So it appears 50 might be the upper limit for GoldenGate.

High-throughput plasmid DNA purification for 3 cents per sample.

(2023-04-12) They basically use a little enzyme cocktail + a microwave(?!) to do high throughput minipreps! Doesn't require beads or anything, and still gives good enough sequencing results. Hype!

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

(2023-04-12) Another 1 step high throughput miniprep protocol. I don't like this one quite as much, since they didn't high throughput the ever-loving-shit out of it, like the above paper. The basic approach is to use NIDs (Non-ionic detergents) to do the lysing instead of a more basic Alkali procedure.

Chance promoter activities illuminate the origins of eukaryotic intergenic transcriptions (2023)

(2023-04-12) Extremely concise in their abstract with the results: "We approach this question by comparing chance promoter activities with the expression levels of intergenic regions in the model eukaryote Saccharomyces cerevisiae. We build a library of over 10^5 strains, each carrying a 120-nucleotide, chromosomally integrated, completely random sequence driving the potential transcription of a barcode. Quantifying the RNA concentration of each barcode in two environments reveals that 41–63% of random sequences have significant, albeit usually low, promoter activities." Their references 25, 26, and 27 are interesting in particular, speaking about yeast promoters.

Engineering artificial photosynthetic life-forms through endosymbiosis (2022)

(2023-04-12) Basically, they get artificial synthetic life forms, but they only last like 20 generations. The way is, well, basically what you'd imagine: cyanos in s cerevisiae. I wonder if there are papers with stable endosymbiosis?

Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology (2022)

(2023-04-12) They basically did the nano-sav-seq primer bind thing. Nice.

OSTIR: open source translation initiation rate prediction (2019)

(2023-04-12) They just fixed Howard Salis's broken RBS calculator. It's still V1, so questionable, especially with newer AI approaches, but man. Thank god SOMEBODY did that.


Benjamin R Gilbert — Modeling Chromosome Replication and Segregation in JCVI syn3A (2022)

(2023-03-30) Part of the minimal cell conference. Basically, the idea was to model replication constrained around ribosomes, winding the simulated DNA around ribosomes, since we can physically see the layout of ribosomes in cells. Quite neat ideas on how to really simulate cells.

Reading list

Thanks to Isaac Larkin for a lot of these suggestions.

Design of protein-binding proteins from the target structure alone

Improving de novo Protein Binder Design with Deep Learning

Noninvasive assessment of gut function using transcriptional recording sentinel cells

PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints

Prediction of protein–ligand binding affinity from sequencing data with interpretable machine learning

A framework to efficiently describe and share reproducible DNA materials and construction protocols

A Well-Characterized Polycistronic-Like Gene Expression System in Yeast

Highly-Automated, High-Throughput Replication of Yeast-based Logic Circuit Design Assessments

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

ColabFold: making protein folding accessible to all

Directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase

Design of typical genes for heterologous gene expression

A red light–responsive photoswitch for deep tissue optogenetics

Enhancing bioreactor arrays for automated measurements and reactive control with ReacSight

Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles

My list:

Integrative illustration of a JCVI-syn3A minimal cell

Current commercialization status of electrowetting-on-dielectric (EWOD) digital microfluidics

Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes

Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool