Safety
There is risk associated with almost any laboratory experiment, especially when working with live biological agents. However, our team has gone to great lengths to minimize the risk posed to the researcher, the public, and the environment. Overall, our project can be considered low risk since the finished product is not intended to come in contact with humans in any form. Our E. coli and Rhodobacter sphaeroides cultures should only be grown and tested in a bioreactor with proper laboratory safety technique. Even in the event that either strain was ingested, its is very likely that no harm would occur since the bacteria are not able to survive the environment of the human digestive system. The two major safety concerns associated with our project arise during DNA purification using gel electrophoresis and extraction of PCB from spirulina powder.
Gel electrophoresis is necessary to purify DNA for almost all steps of our project. To make DNA visible as it moves through the agarose gel, Ethidium Bromide (EtBr) is added to the gel to act as a fluorescent tag. Ethidium Bromide itself is a potent mutagen and a known carcinogen that may be absorbed through the skin. For this reason, a separate lab bench has been set aside for all items coming into contact with the toxin including pipets, tips, gel rigs, glassware, and DC power sources (for electrophoresis). In addition there is a vessel designated as contaminated where all disposables (i.e. pipet tips) maybe deposited and properly disposed of at a later date. It is important that all items coming in contact with EtBr remain on the EtBr lab bench and all noncontaminated lab materials are not brought to this bench. Nitrile gloves must be worn when handling EtBr and replaced frequently, especially when going back and forth from this bench to another one.
Phycocyanobilin (PCB) is necessary for the final testing and characterization of our strain of Rhodobacter sphaeroides and is most easily obtained by purifying it from spirulina, a cyanobacteria. There are two potentially harmful chemicals used in this process: methanol and Mercury (II) Chloride (HgCl2). Methanol is highly flammable and poisonous if ingested. Methanol can cause blindness if swallowed in even small quantities. HgCl2 is a highly toxic form of Mercury because it is in a soluble form and can accumulate in fatty tissues. All steps in PCB extraction must be performed on a designated lab bench in much the same way as gel electrophoresis (although separate from EtBr contamination). A fume hood should also be used with nitrile gloves to prevent human contact. Special decontamination vessels are needed that are large enough to hold all waste products given that there are several washings which produce a large volume of toxins. Mercury requires special disposal procedures that may vary depending on lab protocol.
The general public is at little to no risk from our experimentation since it is not intended for use outside of a bioreactor or for human trials in any way. Our labspace was shared with several classes so it was important that we clean up our lab when finished and that we properly label all chemicals and contamination areas. Environmental procedures were also followed with the disposal of all chemicals, glassware, and sharps. None of our live cultures were released into the environment and therefore would not affect natural populations of bacteria.
All research conducted at Washington University (medical school and main campus) is under the supervision of the Office of the Vice Chancellor for Research (OVCR). This office is responsible for all concerns pertaining to research and publications produced by the university. Among other things, our lab must comply with the Environmental Health and Safety Policy and Procedures laid out by the OVCR. To view the full list of Environmental Health and Safety Policies and Prcedures, please click [http://ehs.wustl.edu/new/safetycommittee.htm here]. Furthermore, every team member has completed an Annual Regulatory and Safety Review for Laboratory Personnel offered by the Environmental Health and Safety committee at Washington University. This review is offered annually and all of our team members have at least one year of lab experience working with live cultures
Our new BioBricks consist of composites of existing BioBricks and new ones derived from the Rhodobacter sphaeroides genome. None of these BioBricks are hazardous or require any special storage procedure beyond that of ordinary DNA fragments.
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Sharing
The following is our Human Practice proposal.
Abstract
We propose a new practice in sharing biological parts whereby it is the duty of the users to consistently report back to the registry on the quality of their part and to make their best effort to replace it in the registry if it is found to be faulty or non-functioning. To encourage this dialogue between the registry and its users, we suggest making it a requirement of iGEM teams that use biological parts to note on the part webpage whether the part worked as expected via a revised rating and review system. Further incentive for user-participation could be achieved through the creation of an additional Gold metal criterion requiring iGEM teams wishing to acquire this award to have replaced a missing or faulty biological part by contacting the initial submitter and restoring the part to a functional biobrick form.
Introduction
The rise of Synthetic Biology over the last decade can be largely attributed to the dedicated community of users who have elected open-disclosure as a means of promulgating this nascent subject. Collaboration and contribution to publicly accessible websites (such as OpenWetWare and the Registry of Standard Biological Parts) have allowed the field to progress at a rate unimaginable prior to the advent of the Internet.
This newfound willingness to disseminate previously coveted methodology and resultant data prior to publication (via online means) mirrors the rise in popularity of social networking websites, such as Facebook and MySpace: social networking users are willing to publically publish extremely personal information. *
A benefit to this type of research design is the ability for researchers to quickly and easily post relevant methodology and data that can be instantly utilized by other scientists worldwide. However, this type of instantaneous submission can often be detrimental to the quality of information being published, which is precisely why a standardized peer-review process is required for publication in a scientific journal, such as Nature.
Peer-review (or “self-policing”) can be obtained in the aforementioned types of delocalized online communities; however, achieving it requires both an immense amount of frequent user involvement and a vast user base with expertise in each area of discussion. Further hindrance to online content quality includes the allowance of anonymity on most online sites as well as an inability to ensure quality of content over time (i.e. if a piece of material previously published remains accurate/reliable in the future). The former challenge raises further doubt as to the validity of user-generated content, while the latter displays a lack of continual quality control of said content.
iGEM’s use of the wiki format and the Registry of Standard Biological Parts is unique in that user’s are required to use these tools over the course of participation in the competition, thereby providing a user-base that actively utilizes the collaborative medium. Furthermore, users are validated prior to being allowed to make any additions or changes, thus resolving the issue of anonymity. However, the ability to maintain quality control remains an issue that is yet to be addressed, particularly with regards to the products in the Registry of Standard Biological Parts.
Accordingly, we propose a change to current method of rating and review as well as an incentive based program to promote consistent participation in the program from the user-base.
- (Note: Obviously, there are numerous other inherent risks to this type of public disclosure in both social networks and Science 2.0 websites. The risk of harm to the individuals providing the information in both the former and latter situation is a fascinating ethical quandary that is oft discussed and written about. However, these ethical problems are beyond the scope of this particular paper and are not applicable to our Human Practice proposal.)
Motivation
Our project hinges on the proper function of Cph8 (BBa_I15010). This synthetic light-sensing protein is composed of a fusion between EnvZ histidine kinase from E. coli and the phytochrome Cph1 from the cyanobacterium Synechocystis. It was first submitted to the Registry of Standard Biological parts by Jeff Tabor (UT Austin) in 2004 and required a silent mutation at base 108 to remove a PstI cutsite to make the part compatible with RFC[10].
It has since become one of the most high profile parts in the registry and is the red light-sensing component of a featured part, the “Light Sensor” ([http://partsregistry.org/Featured_Parts:Light_Sensor]). This light sensor turns a plate of bacteria into biological film where the bacteria in the dark produce LacZ, which converts S-Gal into a black-precipitate, and the bacteria in the light remain the natural color of E. coli. This system was also featured in the November 24, 2005 Nature edition under the title “Engineering Escherichia coli to see Light” (Levskaya et al).
Unfortunately, the copy of this part that is maintained by the registry does not pass the quality control standards. The sequencing of the part does not align properly to the presumed sequence submitted and the gel-cut results using the RFC[10] sites are not as expected. While unfavorable to our iGEM team, this information was much appreciated as it allowed us to move forward without trying to figure out why cloning with Cph8 wasn’t working.
We then contacted Dr. Chris Voight at UCSF, whose lab had originally constructed Cph8, to request the DNA coding sequence in non-biobrick form. Adding Cph8 to our construct required working around the PstI cutsite in the coding region, adding a biobrick prefix and suffix via PCR, and cloning Cph8 into a vector without using PstI. Essentially, we used ad-hoc genetic engineering to continue building our construct, which in retrospect was a poor decision that held back our progress significantly.
As a team we decided that we would not want anyone else to have the same experience working with Cph8 as we had and realized that it was our responsibility to correct Cph8 and replace it in the registry. While we had not succeeded on this front as of the deadline for iGEM 2009 DNA submission to the registry, we plan to carry out site-directed mutagenesis and resubmit the part in the near-future.
Current Design
The Registry of Standard Biological Parts currently has a barebones means of user-review for biobricks in the registry. However, its presence is easy to miss, as the only evidence of it appears at the very bottom of the biobrick main page and consists solely of a “sample” review box.
Though the sample review box appears to be useful upon initial inspection, clicking on the image brings the user to the “Experience” page, wherein there resides nothing that even remotely resembles the “sample” image that leads the user there to begin with. Furthermore, neither the main biobrick page nor the “Experience” page provides any type of interactive user-interface that would even slightly entice the user to submit a review. In fact, if the user does want to submit any sort of review on the biobrick, they must enter their review in the same wiki text box that is used to edit content pages and the user must also include all appropriate code for formatting, for which there is no immediately apparent and available template. It is precisely this type of constraint that has restricted the utilization of the nascent review tool and is the culprit for the vast number of antiquated biobricks, which are consequently useless to any user interested in acquiring the part.
It is important to note, however, that the mere existence of the “sample” review image and “Experience” page verifies our belief that there is indeed a communal need for this type of tool and at least an attempt is being made by the developers to address this need. Accordingly, our proposed design will build off of this current framework so as to transform a currently ineffective tool into an invaluable resource for all Registry users.
Proposed Design Changes
- Proposed design changes will be here shortly!
Conclusion
- Conclusion will be here shortly!
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