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Project Description
Construction of a light and oxygen sensor to control protein expression regulation
Rhodobacter sphaeroides (Rhb. sphaeroides) is a purple non-sulphur bacterium that belongs to the alpha-proteobacteria, the most metabolically diverse group of organisms on the planet. Part of this versatility is attributed to regulatory proteins which coordinate different metabolic states such as anaerobic photosynthesis and chemi-heterotrophy.
Specifically, our project is concerned with two regulatory protein systems. The first one, is the two-component global activator under anaerobiosis, PrrB/PrrA. The second system is the light and oxygen mediated repressor/anti-repressor PpsR/AppA. These act in conjunction, along with a slew of other proteins in Rhb. sphaeroides, to tightly control and balance the metabolic demands of the cell. Our team will take these two regulatory systems to express them into a Rhodopseudomonas palustris chassis. The goal is to achieve a better comprehension of the R. palustris regulatory networks through the study of their properties of genetic switches in expressed in a relative isolation within another organism, considering the interference caused by other proteins could be minimal. This will allow us to study in a more precise way how the regulatory systems sense external conditions and transduce them into alternative expression levels via signaling pathways.
For us to achieve this goal a cassette in which GFP expression is light-dependent by the antirepression of PpsR and oxygen dependent by the activation of PrrA/B system has been designed. All this lab work is accompanied by a computational model which, based on our experimental data, will provide a way of testing our knowledge of these systems. This in turn allows us to enhance the functionality of the device as it expresses heterologous genes in R. palustris.
Once we have characterized the functionality of these regulatory networks we aim to take advantage of R. palustri’s metabolic versatility, and use this functional bacteria as a microbial factory that will allow the production of products of economic value from byproducts otherwise considered pollutants such as CO2 and other industrial derivatives.
Safety
Question 1:'''Would any of your project ideas raise safety issues in terms of researcher, public or enviromental safety?'''
Our intention is to re-design two regulation systems from Rhodobacter sphaeroides, and introduce them into Rhodopseudomonas palustris. Since the PCR obtention of the 5 proteins that form the system is extremely difficult, we use the gene synthesis technology provided by Genescript in order to get this genes. Purple Non sulfur Bacteria are one of the most metabolically diverse groups known in nature, they are able to grow under different conditions such as, aerobic respiration, anoxigenic photosynthesis, anaerobic fermentation. Due to this ability, plus their ability to eat aromatic compounds, these bacteria can be found in hostile media such as polluted water. Despite all these features, work with these microorganisms in lab conditions is very hard, its growth medium is composed by a complex mix of metals and vitamins. We can consider that the risk in PNSB management in the lab is very low since they are non-pathogenic bacteria. R. sphaeroides and R. palustris´s grow rate is quite slow, thus, our genetic systems do not represent a public safety menace since they are highly specific, we have designed this systems for its specific functioning in purple photosynthetic bacteria. Nevertheless is very important to take special care when working in the wetlab, we have to take into consideration the basic precautions in microbiology.
Question 2: '''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''
No, our BioBricks parts come from Rhodobacter sphaeroides, this bacteria is non-pathogenic and it can be worked in a Biosafety level 1 laboratory, based on the CDC Biosafety in Microbiological and Biomedical Laboratories (CDC, 2007). The functionality of our parts do not produce any dangerous compound, we are working in control of gene expression. Furthermore, we used BioBricks from iGEM distribution for all our assemblies, for example GFP as reporter, these parts do not represent a biological risk.
Question 3: '''Is there a local biosafety group, committee, or review board at your institution? If no, which specific biosafety rules or guidelines do you have to consider in your country?'''
A Biosafety committee would have to be integrated by a multidisciplinary group of members among Institutions and Universities and it should provide expert advice on laboratory, biosafety and biosecurity issues related to research and other academic activities. Plan, design and implement the necessary instruments for biosafety risk assessment. Establish, disseminate and analyze safety measures in handling chemicals, genetically modified organisms and potentially infectious pathogens that will allow to make timely decisions for users in order to minimize potential health risks. Conduct briefings and feedback sessions with the areas involved in biosafety. Biosecurity measures established in the regulations must be consistent with the development and promotion of basic and applied research. For the analysis of solutions to particular problems should be evaluated case by case, the benefits and potential risks of the use of GMOs, this analysis should also include assessment of the risks of alternative technological options for coping with the specific problem to which the GMO was designed. Encourage and ensure the mechanisms that establish responsibilities who violates the regulations under existing legislation. And finally,it is important that other laws are reviewed; this will help to strengthen biosecurity aspects of other organisms that are not GMOs.
Question 4: '''Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''
As a part of our human practices work, we are trying to implement an administrative tool in order to answer the most important questions in terms of safety and biosecurity, this tool is the Quality Function Deployment. The relevance of this work is that we can be able to use the results to make different proposals that could support the development of synthetic biology based projects in Mexico.
References
Biosafety in Microbiological and Biomedical Laboratories, (BMBL) 5th Edition (HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health; U.S. Government Printing Office: Washington DC; 2007).
