Team:Virginia/Project

Genetic Attenuator

Transcriptional attenuation is a very promising new tool for the specification and control of genetic transcription rates. The basic mechanism is simple: stop some fraction of the polymerases before they reach the desired gene to reduce transcription (and thereby translation) of the gene. Natural terminators already serve this role in their capacity to prevent transcription of undesired DNA, but a heretofore untapped resource is the potential inefficiency of this termination. For instance, imagine a terminator that interrupts only 50% of the polymerases transcribing it. If placed between two genes, this will result in 100% more copies of mRNA corresponding to the first gene than the second in the total transcript output. Without any sophisticated empirical testing and tuning (as is often required for promoter engineering), using a tool directly out of the BioBrick toolbox, an accurate and reliable ratio of gene transcription can be established.

BioBrick Placeholders

A new technical standard!

Although standardized, assembling composite BioBricks from basic parts has its limitations. For example, parts are added to the ends of a BioBrick. In other words, there is no easy way to insert a new part within an already constructed BioBrick. That is, until now! We've developed a new technical standard called BioBrick Placeholders. Simple and elegant, these BioBricks serve as placeholders by providing unique but compatible multiple cloning sites. BioBrick Placeholders are simply BioBrick-compatible restriction sites flanked by the standard BioBrick prefix and suffix. Next time you go to build a gene but you're not sure which RBS to use, go ahead and put in a BioBrick Placeholder to reserve a spot for that RBS. Later, after you've amplified your construct, you can insert any RBS you'd like.

The following 8 BioBrick Placeholders have been submitted to the Registry. These parts include compatible restriction sites as listed in "Engineering BioBrick Vectors from BioBrick parts."

1. EX - ApoI (NotI) AvrII - SP
2. EX - ApoI (NotI) NheI - SP
3. EX - ApoI (NotI) NsiI - SP
4. EX - ApoI (NotI) SbfI - SP
5. EX - MfeI (NotI) AvrII - SP
6. EX - MfeI (NotI) NheI - SP
7. EX - MfeI (NotI) Nsil - SP
8. EX - MfeI (NotI) SbfI - SP

BioPlastic

Making plastic a renewable resource.

Rastonia eutropha naturally produces PHB for long-term carbon storage. Taking advantage of this microbes metabolic abilities, we've codon-optimized the 3 essential genes that code for the PHB biosynthesis pathway for expression in E. coli. The enzymes that are produced are PhaA, PhaB1 and PhaC1, which take acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and 3-hydroxybutyryl-CoA to poly-3-hydroxybutyrate (PHB), respectively. Why bother synthesizing PHB? It has the potential to replace polypropylene, a petroleum derivative, as main component of plastic materials.

Adding to the Registry

More tools in the toolbox.

Over the course of our research we identified a few key areas in the registry where we felt it was lacking. Namely we saw a dearth of reporters as well as a limited number of anti-biotic resistances to work with. Expanding the scope of the registry is a key aspect of iGEM and we feel glad to contribute.

Reporters are a staple of modern biology. This year's Nobel Prize in Chemistry was awarded for the discovery of GFP. Reporters such as GFP allow the visualization and monitoring of synthetic biological systems. However, there are only a handful of fluorescent proteins available in the Registry at this time. Diversity in reporters allowed projects such as Brainbow to be created. We have thus added two new reporters to the registry: Strongly Enhanced Blue Fluorescent Protein (SBFP2) and Orange Fluorescent Protein (OFP).

OFP in action. As synthetic biological systems become more complex, there will be a need for many more FPs and, eventually, better ways to visualize what's going on inside the cell.

We also introduce a new antibiotic resistance (to streptomycin) to allow more flexibility and diversity in plasmid construction (aadA).