Engineered Microbes Clean Up Copper & DNA Cloning Goes Full-Auto
By: Niko Mccarty
Tags: Science, Tech, Research, Newsletter, News

NEWSLETTER

Engineered Microbes Clean Up Copper & DNA Cloning Goes Full-Auto

This Week in Synthetic Biology (Issue #17)

This Thanksgiving, I’m grateful to you, my readers. In the convoluted chaos of the internet, I know that you could be reading many things. Thank you for reading this. Reach out on Twitter with feedback and questions.

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DNA Cloning Goes Full-Auto

It’s midnight in the lab. All your co-workers have gone home. You’re alone at the bench, fearful that a security guard will arrive at any moment and kick you out. If you, like me, have found yourself in this situation, it may be because you have failed — repeatedly — to clone a particularly tricky DNA sequence.

But that could happen a lot less in the near future. Robots are coming online, promising to automate entire experiments. So, too, could robots be used to create and clone custom DNA sequences. Unfortunately, a specific step in the cloning process — blasting cells with electricity to coax them into taking up DNA — has proven troublesome for machines.

A new study, published Nov. 24 in ACS Synthetic Biology, offers a fully automated protocol for DNA cloning that relies on “natural transformation”, rather than electroporation. Sang Yup Lee’s lab, at the Korea Advanced Institute of Science and Technology, demonstrated that Acinetobacter baylyi, a type of bacteria, efficiently take up DNA, without the need for specialized equipment. Researchers already knew that A. baylyi were “naturally competent”, but I think this is the first time that these bacteria have been incorporated into a fully automatic, DNA cloning pipeline.

The team simply dropped DNA sequences into liquid with A. baylyi, and the cells gobbled them up and began to reproduce, making tens, then hundreds, then thousands of copies of the DNA.

“No DNA purification, competence induction, or special equipment is required,” the authors write. “Up to 10,000 colonies were obtained per microgram of DNA, while the number of false positive colonies was low.” The team used the new protocol to clone 21 biosynthetic gene clusters, with lengths ranging from 1.5 to 19 kb, and showed that the protocol was relatively consistent when performed by an Opentrons robot. Check out the paper! Link

Engineered Bacteria Clean Up Copper

A team of researchers at the University of York and Umeå University, in Sweden, have engineered E. coli bacteria to accumulate heaps of copper.

To do that, the team fused seven different snippets of proteins — each thought to bind copper ions — to a protein called Maltose Binding Protein, or MBP. These protein chimeras, when expressed inside of the bacterial cells, “conferred tolerance to high concentrations of copper sulphate,” write the authors. The tolerance to copper was so high, in fact, that some bacteria could withstand concentrations “160-fold higher than the recognised EC50 toxic levels of copper in soils.”

The researchers also crunched some data, on computers, and found that these copper-binding proteins might be able to bind other types of metals. The authors suggest that these bacteria could “be adapted for the removal of other hazardous heavy metals or the bio-mining of rare metals.” Let’s mine some asteroids with juiced up space bacteria!

This work was published in Scientific Reports, and is open access. Link

Knocked Down Genes Reveal How Metabolism Heals

Drop a bacterium into a new environment — one with more sugar, or different competitors — and its metabolism will quickly adapt. In fact, the metabolism of a bacterium can change so much, when placed in a new environment, that the total mass of its various enzymes can double, according to a 2016 study by Schmidt et al. in Nature Biotechnology.

Okay, so lots of protein levels change when a cell feels stressed out. But what happens when just one gene, encoding one enzyme, is repressed, or knocked down? How does the rest of the cell’s intricate metabolism shift in response to account for that single, “deficient” gene?

It turns out that cells shift their cellular resources in intriguing ways to “heal” a broken link in the metabolism chain. A new study, published Nov. 24 in Cell Systems, used CRISPRi (CRISPR interference) to repress one gene at a time in E. coli cells. Researchers used an inducible version of CRISPRi that can be turned ‘on’ at will “to investigate how E. coli metabolism responds to decreases of enzyme levels.”

For the study, the team repressed 1,515 different genes involved in metabolism, creating 4–6 sgRNAs for each gene; that resulted in a total of 7,177 unique strains of E. coli. They then “induced” the CRISPRi system, and measured “the time delay between inducer addition and appearance of fitness defects.”

30 of the strains were studied in greater detail, unveiling some interesting examples of metabolisms “adjusting” to account for a deficient enzyme in a pathway. “Overall, our results highlight the central role of regulatory metabolites in maintaining robustness against ever-changing concentrations of enzymes in a cell,” the authors wrote.

To learn a bit more about this study (in general language), check out the press release. This study is open access. Link

Bacteria Evolved in Lab to Study Antibacterial Resistance

Drug-resistant bacteria are a growing issue, and researchers are racing to develop new antibiotics to fight back. To make better antibacterial compounds, scientists must first understand how antibacterial resistance emerges.

Published Nov. 24 in Nature Communications, a team — from RIKEN and the University of Tokyo — used an automated, robotic system to repeatedly passage bacteria over more than 250 generations, while grown in the presence of 95 different antibacterial chemicals. In other words, they put cells into evolutionary overdrive.

The researchers analyzed the evolved cells, studying how gene expression profiles changed for each strain, and then fed their massive dataset into a machine learning algorithm. They identified several gene expression “signatures” that were associated with drug resistance in the microbes. Read the press release on this open access study. Link

Super Accurate Insertion of DNA Chunks with CRISPRi

A special type of CRISPR-Cas system, harnessed from Vibrio cholerae bacteria, enabled researchers to integrate large chunks of DNA, up to 10,000 bases in length, at specific sites in the genome of bacteria. The method is supposedly 100 percent efficient.

The team, from Columbia University, call their method INTEGRATE (insertion of transposable elements by guide RNA–assisted targeting). After demonstrating their method by inserting DNA at one location in the genome, they scaled up their work in a massive way: By expressing multiple guide RNAs in the cells, the team was able to insert DNA chunks at three places in the genome at once. This work was published in Nature Biotechnology. Link