Hot off the press

Hot off the Press highlights recently published work for the benefit of our readers. Our contributors this month have focused on the synthesis of the complete genome of bacterium Mycoplasma genitalium. New contributors are always welcome. If you are interested please contact molbiosyst@rsc.org for more information, we'd like to hear from you.

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One large step for man…

Reviewed by: Sachdev Sidhu and Andreas Ernst, Genentech Inc., CA, USA

 

Over the past decade, DNA synthesis technology has improved to the point where the assembly of whole genes has become trivial. This has opened up the possibility of synthesising whole chromosomes and genomes, which would be the first of many steps to creating artificial life. It now appears that researchers at the J. Craig Venter Institute have achieved this goal, at least on the scale of the simple bacterium Mycoplasma genitalium.

Smith and co-workers describe the assembly and cloning of an approximately 600 kb synthetic genome, named M. genitalium JCVI-1.0, from chemically synthesised building blocks. This is so far the largest assembly of synthetic DNA and opens the door to manipulation of DNA on a whole genome level. The authors solved the complex task of genome building by a combination of in vitro assembly of small fragments and in vivo assembly of larger fragments in yeast. The genome was split into approximately 100 fragments, which were chemically synthesised with small overlaps with adjacent fragments. In the next step, sticky ends were generated with an exonuclease enzyme and these were used to anneal and stitch together four adjacent fragments at a time. The incidental gaps were repaired with additional enzymatic treatments, resulting in the in vitro assembly of 25 bacterial artificial chromosomes of approximately 24 kb each. These steps were repeated to eventually obtain four “1/4 genome” fragments of approximately 150 kb each. At this stage, the authors found that the stability of the artificial chromosome could not be maintained in Escherichia coli and they switched to yeast. The last two assembly steps were carried out by constructing circular yeast artificial chromosomes containing centromeres to ensure genetic stability. To verify the success of their assembly, they introduced specific “watermark” sequences, which enabled confirmation of the origin of the DNA.

This report represents an important step for the field of synthetic biology and, in itself, the paper is a remarkable technical tour de force. However, the work can best be considered as a first fragment of a larger puzzle, as it doesn’t yet answer or pose significant questions beyond the ever-present technical question, “Can we do this?” With the new methodology, it will be possible to generate libraries on the genomic scale, but of course, the significant problem of translating the genome to a functional proteome remains unsolved. If techniques can be developed for introducing synthetic genomes into an environment where they can give rise to a functional cell, the possibilities for novel research and enquiry will indeed be endless.

Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, Jayshree Zaveri, Timothy B. Stockwell, Anushka Brownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III, Hamilton O. Smith, Science, 2008, DOI: 10.1126/science.1151721

Hot off the RSC press

Genetic testing in a shoe-box

Reviewed by: Gavin Armstrong, Royal Society of Chemistry, UK

 

Canadian scientists have succeeded in building the least expensive portable device for rapid genetic testing ever made.

The cost of carrying out a single genetic test currently varies from hundreds to thousands of pounds, and the wait for results can be weeks. Now a group led by Christopher Backhouse, University of Alberta, Edmonton, has developed a re-useable microchip-based system that costs just £500 to build, is small enough to be portable, and can be used for point-of-care medical testing (Fig. 1).

Fig. 1 The device is about the size of a shoe-box with the optics and supporting electronics filling the space around the microchip.

The well-known techniques reverse transcription, polymerase chain reaction and capillary electrophoresis have been developed over recent decades to take tiny amounts of genetic material and grow and amplify them. These handling techniques make detecting genes possible, and have previously been miniaturised so they fit on a microchip that uses small channels, valves and reaction chambers.

The team has redesigned the gene handling microchip, and used a different detection method, to develop a system that is ‘comparable in performance to much bigger and more expensive machines,’ explained Backhouse. The size of the device is reduced to that of a shoe-box with optics instrumentation and supporting electronics filling the space around the microchip.

To keep costs down, ‘instead of using the very expensive confocal optics systems currently used in these types of devices we used a consumer-grade digital camera,’ Backhouse explained.

The device can be adapted for use in many different genetic tests. ‘By making small changes to the system you could test for a person’s predisposition to cancer, carry out pharmacogenetic tests for adverse drug reactions or even test for pathogens in a water supply,’ said Backhouse.

The group strives to make genetic testing accessible to everyone in the same way computers are now. ‘It’s not long ago that computers were inaccessible to most people but now we all carry more than one on our person. This was made possible by integration and cost reductions,’ said Backhouse. He said he plans to cut the manufacturing costs of this device to £50 in the very near future by integrating more of the electronics and further miniaturising the microfluidics.

Govind V. Kaigala, Viet N. Hoang, Alex Stickel, Jana Lauzon, Dammika Manage, Linda M. Pilarski and Christopher J. Backhouse, Analyst, 2008, DOI: 10.1039/b714308g

A logical approach to designer cells

Reviewed by: Kathleen Too, Royal Society of Chemistry, UK

 

Logic gates made from proteins could lead the way to yeast cells that can self regulate during fermentation, say US scientists.

A team led by Virginia Cornish at Columbia University, New York, has designed a transcription factor that functions as an AND logic gate. Transcription factors are proteins that form part of the system controlling the transfer (transcription) of genetic information from DNA to RNA. It usually possesses a DNA binding domain and an activation domain. When both domains are turned on, transcription occurs. This gate forms part of an artificial transcription factor network for programming functions in a cell. Cornish explained that the networks are designed ‘like electrical circuits with transcription factors functioning as Boolean logic gates.’

Using a well-defined yeast system, the US researchers genetically separated the DNA-binding domain and the activation domain so that transcription only occurs in the presence of a small molecule ligand (Dex–Mtx). On one end of the small molecule ligand is dexamethasone (Dex) which has high affinity for the activation domain whilst at the other end of the ligand is methotrexate (Mtx) which binds to the DNA-binding domain. Dex and Mtx are held together by a carbon linker. In this system, the DNA-binding domain, the activation domain and the ligand (Dex–Mtx) form the three inputs of the logic gate. When all the three inputs are turned on, the output is transcription (Fig. 2). This is analogous to a three-input AND gate.

Fig. 2 All three components of the transcription system are needed to switch transcription on.

Nicholas Buchler, an expert in the field at Rockefeller University, New York, US, said that this will ‘greatly benefit’ research into engineering cells capable of making small molecules. Jonathan Bronson, who works with Cornish, suggested the technology could be used to ‘create yeast cells that could monitor conditions in a fermentor and adjust themselves to optimise ethanol/glycerol production.’

Ron Weiss, a specialist in cell programming using logic circuitry at Princeton University, US, said that he hopes that this ‘system can be adapted to different proteins inside the cells, creating multiple versions of AND logic gates.’

Bronson plans to go one step further and obtain ‘a toolkit of transcription factors capable of performing many types of logic gates, like NAND, that could be used to create any genetic circuit that we can imagine inside a microbe.’

Jonathan E. Bronson, William W. Mazur and Virginia W. Cornish, Mol. BioSyst., 2008, 4, 56, DOI: 10.1039/b713852k

Lanthanide ions hold key to disease screening

Reviewed by: David Barden, Royal Society of Chemistry, UK

 

Canadian researchers have devised a way to assess biological samples for the presence of multiple small molecules, which has profound implications for the rapid identification of diseases.

Being able to distinguish diseased cells from healthy ones is vital in the identification of diseases in humans. A rapidly advancing way of doing this is to determine the concentrations of small molecules (biomarkers) present in biological samples, as amounts of these chemicals differ between diseased and healthy cells. Vladimir Baranov and colleagues from the University of Toronto have now developed a sensitive method that, by using lanthanide ions, is able to determine the concentrations of many biomarkers at once. This, they say, will have important applications in clinical diagnostics.

Baranov and colleagues bound ¹⁵¹Eu ions to a polymer chain chemically attached to an antibody, which itself binds to a natural biomarker (Fig. 3). Once the unreacted antibody derivative is washed away, the sample is exposed to inductively coupled plasma mass spectrometry. This atomises the entire sample and allows the elemental composition to be determined. In this case, the amount of ¹⁵¹Eu correlates to the amount of biomarker present in the original sample. The number of ¹⁵¹Eu ions within each molecule means that the method is more sensitive to the amount of biomarker than existing techniques, said Baranov.

Fig. 3 A schematic showing the lanthanide tagging of antibodies with a background of cancer cells.

The power of the team’s method is that it can measure many biomarkers simultaneously, simply by using a different one of the 50-plus stable lanthanide isotopes for antibodies against different targets. Baranov’s team is now working to apply the technique to the classification of leukaemia samples.

The importance of this research is emphasised by Les Ebdon, Vice-Chancellor of the University of Bedfordshire, UK, who said ‘This exciting study promises to advance our abilities to understand and diagnose complex diseases such as leukaemia.’

Olga I. Ornatsky, Robert Kinach, Dmitry R. Bandura, Xudong Lou, Scott D. Tanner, Vladimir I. Baranov, Mark Nitz and Mitchell A. Winnik, J. Anal. At. Spectrom., 2008, DOI: 10.1039/b710510j

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