A Luminsescent Biosensor with Increased Dynamic Range for Intracellular cAMP

We had an interesting talk about biosensors recently, allowing live cell imaging of important biochemicals. I think one of the major problems that re-occurred was the lack of dynamic range. So, there wasn't enough of a change in fluoresence to be a really useful tool, even though the binding component was able to selectively bind its ligand and induce conformational changes. Here, Promega has developed a cAMP biosensor with increased dynamic range. I don't think there are chemical indicators for cAMP. Some additional benefits of using a protein are that you don't have to worry about diffusion out of the cell. This makes FACS possible - cell sorting - allowing you to collect cAMP-sensing cells and compare them no non-cAMP-sensing cells to help get at the biological significance. -That's not in the paper, but I imagine that could be a good future direction to go in.

Site-Specific Incorporation of Photo-Cross-Linker and Bioorthogonal Amino Acids into Enteric Bacterial Pathogens

In this article the unnatural amino acid mutagenesis was used to investigate the acid-survival strategy and the pathogenesis mechanisms of some enteric bacterial species. They reported the incorporation of a photo-cross linking amino acid into a Shigella acid chaperone shHdeA and an amino acid bearing an azido group was incorporated into a Shigella type 3 secretion effector OspF. This is the first time that UAAs were utilized to investigate proteins involved in the pathogenesis and stress-defense mechanisms of virulent Gram-negative bacteria.

A current problem in biomolecular computing has to do with the varying copy numbers of gene products and plasmid stability. The Ying et al paper included here offers an alternate approach. Instead, they propose to integrate the circuitry into the genome itself. This approach confers stability to the circuitry so it can be reliably propagated during cell division, and gives a predictable number of gene products depending on the number of integrated parts in the genome. The authors accomplish this by encoding the entire synthetic network on a plasmid, PCR amplifying it, and then purifying it and transforming it into the competent E. coli cell by electroporation. As the author reports, this refined method leads to reduced leaky expression and predictable copy number. The ultimate goal, an incorporated toggle switch, performs well with distinct outputs and reduced noise.

Essence of life: essential genes of minimal genomes

This is one of the most recent papers about constructing a minimal genome. It goes in depth on how minimal genomes are created (see figure attached), and it also brings up important problems associated with this project. One problem that I thought was interesting was that although they are creating the minimal genome for Mycoplasmas,the genes associated with this cell are not necessarily universally essential.

Ribosomal Route to small molecule diversity

This is an interesting article about engineering the Ribosomal peptide pathway, that makes different kind of natural products, to include unnatural amino acids. Specifically, they are interested in making new types of cyanobactins. The need to diversify macrocycles is important for the drug and pharmaceutical industries. Though this is novel, the synthesis of these peptides may be just as easy. A enzyme at the end could just cyclize the peptide. That would be similar to the NRPS work we have seen in Gavin's class. This technology seems very useful for a known drug or scaffold that needs to be massed produced. I sometimes forget that UAA incorporation can be used for more than just large proteins.

A biomimetic polyketide-inspired approach to small-molecule ligand discovery

In this paper C. Aquino and co-workers designed polyketide derived oligomers called ‘chirlal oligomers of pentenoic amides’ (COPA) by using the split pool technique. They were able to make a diverse set of COPA library and screened these compounds against the DNA binding domain of p53 which is an important transcription factor that regulates cell cycle and apoptosis. It was interesting that they  increased conformational constraints for the small molecules for a higher affinity towards target proteins.  

RNA-Selective, Live Cell Imaging Probes for Studying Nuclear Structure and Function

This paper from the Chang group is one of multiple that I found as a follow up to the paper we read for class today concerning the styryl scaffold. It seems that they did use different building block "A" molecules. They were also looking for RNA specific molecules.

A Genetic Circuit System Based on Quorum Sensing Signaling for Directed Evolution of Quorum-Quenching Enzymes

This paper utilizes quarom sensing circuitry for the directed evolution of AHL-quenching enzymes. Acyl-homoserine lactones (AHL) are a class of diffusible molecules used in bacterial intercellular communication. It is expressed during periods of stress to a bacteria's host organism as an intercellular signal leading to increased virulences when the host's immune response system is weakened. The goal of the authors is to use directed evolution of AHL-quenching enzymes to control AHL concentrations. Through directed evolution two evolved enzymes are produced with increased kcat/kM values, due mainly to increased substrate sensitivity. The screening method used conferred antibiotic resistance to enzymes able to efficiently quench AHL molecules. This study may be used as a template for further quarom circuitry probing and the evolution of enzymes involved.

A Class of Human Proteins that Deliver Functional Proteins into Mammalian Cells In Vitro and In Vivo

Prior studies have shown that the higher the net positive charge a protein has, the more likely it is able to cross the cell membrane. In this article the authors search the human proteome for proteins with the highest positive theoretical charge to molecular weight ratio that could be expressed in E. coli. These proteins, naturally supercharged human proteins (NSHPs), were then fused to reporter constructs and were shown to be able to cross the cell and nuclear membrane of varying cell types. In the figure below, the NSHPs (the six structures shown) and proteins known to penetrate the cell membrane (bottom 3 and +36 GFP) are fused to mCherry and incubated with 3 cell types. The median fluorescence is then determined by flow cytometry.

A β-(1,2)-Glycosynthase and an Attempted Selection Method for the Directed Evolution of Glycosynthases

This article expounds on the topics of directed evolution and development of selection and screening methods. Here, Jakeman et al. employ a strategy for the in vivo directed evolution of glycosynthetases. Glycosynthetases are glycosidases that aid in the synthesis if glycosidic linkages and are designed by mutation of the catalytic nucleophile in a glycosidase. This strategy hinges on the ability to identify active mutant enzyme catalysts. They were able to develop the first glycosynthetase capable of glycosylating amino sugar acceptor substrates with a β-(1,2) glycosidic linkage. This should allow for the solubility enhancement and selective targeting of pharmaceuticals.

Chemical synthesis of bacteriophage g4.

This paper illustrates how an entire bacteriophage genome can be synthesized and sequenced with good efficiency for bacteria infection. This is exciting for many reasons one being the ability to create an organism with an entirely synthetic genome by whole genome assembly. This study has implications for the medical treatment and/or prevention of human disease and further illuminates the possibilities for synthetic biology applications.