Project

The goal of Waterloo's 2011 iGEM project is to implement self-excising ribozymes (introns) as biobricks. But first, what are self-excising ribozymes? Ribozymes are ribonucleic acid (RNA) enzymes and enzymes are reaction catalysts. So ribozymes are just RNA sequences that catalyze a (trans-esterification) reaction to remove itself from the rest of the RNA sequence. Essentially these are considered introns, which are intragenic regions spliced from mRNA to produce mature RNA with a continuous exon (coding region) sequence. Self-excising introns/ribozymes consist of type I and II introns. They are considered self-splicing because they do not require proteins to intitialize the reaction. Therefore, by understanding the sequences and structure of these self-excising introns and making them useable, we can use them as tools to make other experiments easier.

1.0 INTRODUCTION

This design provides a reasonable basis to implement in vivo applications involving RNA level regulatory sequences such as recombination sites. Since recombination sites can interrupt the functional production of a protein, the incorporated ribozyme portions can remove them before the translation phase of gene expression. Functional synthetic antibodies and plants capable of producing domain-shuffled Cry toxins are possible applications. This experimental investigation is a novel tool for producing a wide variety of these compounds with a supplementary regulation step.

1.1 A Little Bit About Group 1 Introns

All group I introns in bacteria have presently been shown to self-splice (with few exceptions) and maintain a conserved secondary structure comprised of a paired element which uses a guanosine (GMP, GDP or GTP) cofactor. Conversely, only a small portion of group II introns have been verified as ribozymes (they are not related to group I introns) and generally have too many regulators to easily work with. It is mainly the structural similarity of these introns that designates them to group II. We will mainly be working with group I introns, such as the phage twort.ORF143.


Group I introns contain a conserved core region consisting of two helical domains (P4–P6 and P3–P7). Recent studies have demonstrated that the elements required for catalysis are mostly in the P3 to P7 domain. They are ribozymes that consecutively catalyze two trans-esterification reactions that remove themselves from the precursor RNA and ligate the flanking exons. They consist of a universally conserved core region and subgroup-specific peripheral regions, which are not essential for catalysis but are known to cause a reduction in catalytic efficiency if removed. To compensate for this, a high concentration of magnesium ions, spermidine or other chemicals that stabilize RNA structures can be added. Thus, the peripheral regions likely stabilize the structure of the conserved core region, which is essential for catalysis.

1.2 Trans-Esterification Reactions

The secondary structures, such as P6, formed by group I introns facilitates base pairing between the 5' end of the intron and the 3' end of the exon, as well as generates an internal guide sequence. Additionally, there is a pocket produced to encourage binding of the Guanosine cofactor. The Guanine nucleotide is placed on the first nucleotide of the intron. The 3'OH of Guanosine group nucleophilically attacks and cleaves the bond between the last nucleotide of the first exon and the first nucleotide of the 5' end of the intron; concurrently, trans-esterification occurs between the 3'OH and the 5'phosphorous from the 5' end of the intron. Subsequent conformational rearrangements ensure that the 3'OH of the first exon is placed in proximity of the 3' splice site. In this way, further trans-esterification reactions and splicing occurs.

1.3 Fusion Proteins

Fusion proteins are combined forms of smaller protein subunits and are normally constructed at the DNA level by ligating portions of coding regions. A simple construction of traditional fusion protein involves inserting the target gene into a region of the cloned host gene. However, the subsequent project design, in its simple construction, interrupts the cloned protein with ribozyme sequences flanking a stop codon. The method proposed deals with excision and ligation at the RNA level, therefore, the unaltered DNA sequence does not code for a functional protein. The ligation of protein coding sequences can create functional fusion proteins for many applications including antibody or pesticide production; however, this method of production is limited to producing the same fusion protein each time since the sequence is not modified in between the transcription and translation phases of gene expression. One disadvantage of this is the resultant resistance of a pathogen to antibodies or a target organism to pesticides. For example, a specific pesticide (Cry toxin) may eventually not be effective to its target plant if subsequent plant generations inhibit its uptake, overproduce the sensitive antigen protein so that normal cellular function persists, reduce the ability of this protein to bind to the pesticide or metabolically inactivates the herbicide. Similar mechanisms contribute to antibiotic resistance. Any resistant organisms will inevitably prevail in subsequent generations. Recombination sites could potentially be incorporated into the subsequent project design to circumvent some of the difficulties with traditional fusion proteins as a result of host resistance. However, recombination sites may interrupt the functional fusion protein from forming. Ribozyme segments at the RNA regulation level can potentially remove disrupting sequence after such shuffling occurs. Therefore, the intervening sequence maintains its DNA level functionality but is removed when no longer needed at the RNA level. Fusion protein design focused on the DNA level does not have this dynamic regulation. 

1.4 The Cre-Lox System

In bacteriophage P1 exists the cre enzyme and recognition sites called lox P sites. This viral recombination system functions to excise a particular DNA sequence by flanking lox P sites and introduce the cre enzyme when the target is to be excised. The cre enzyme both cuts at the lox P site and ligates the remaining sequences together. The excised DNA is then degraded. This is similar to our project design; however, instead of requiring the addition of an enzyme at the desired excision time, the self-excising nature of ribozymes automatically functions during the normal process of gene expression (RNA level). 

2.0 PROJECT IN DETAILS

2.1 EXPERIMENTAL DESIGN

Our protocol will involves the insertion of a functional protein, split by the self-removing elements, between CUCUUAGU and AAUAAGAG in the P6 region of twort.ORF143. GFP (green fluorescent protein) is split into two parts, which will be referred to as GFP1 and GFP2. With a constitutive promoter, GFP1 and GFP2 will be separated by a class 1 A2 intron split into two (for now, IN1 and IN2) sequences that flank another sequence inserted into the P6 loop, which was chosen because anything attached to this region will remain outside the protein. Note that this experimental design also contains an in frame stop codon, which is expected to be spliced out of the sequence with IN1 and IN2 and will utilize the RFC53 convention. Following GFP2 is a transcriptional terminator (TT). The method of making this construct is detailed in RFC53. Below is Figure 1 through Figure 3. They illustrate the order of parts in the design and the trans-esterification reaction that results in a function GFP:

2.2 CONSTRUCTION MAPs AND RFC 53

As per RFC 53 convention, enzyme digestions are followed in the particular order outlined below. The standard procedure makes this technique reproducible, therefore, more easily extrapolated to other applications. Compared to other protein fusion methods, this design facilitates additional regulation within necessary guidelines. However, the embedded post-transcriptional modification in this design is a complication to consider in simpler designs where regulation at this level is not necessary. As such, unnecessary bulk in plasmid vectors is known to add to metabolic load.

2.2.1 General Construction Map

The following figure graphically shows the laboratory procedure for the experimental design in the form of an enzyme map:

K576005 contains the first component of GFP (GFP1)

K576003 contains the first part of the intron sequence (IN1

J61046 contains the lox site

K576006 contains the second component of GFP (GFP2)

K576004 contains the second part of the intron sequence (IN2)

K576007 contains GFP1 and IN1

K576009 contains GFP1, IN1 and lox1

contains the promoter (P), ribosomal binding site (RBS), GFP1, IN1, lox site, IN2, GFP2 and transcriptional terminator (TT). This is the final construct (experimental design).

2.1.2 Controls' Construction Map

Controls are necessary to prove that the design of this experimental investigation is functional and more practically for comparison of fluorescence in the laboratory. In the positive control, GFP1 and GFP2 flank either RFC25 or RFC53, which will not disrupt translation regardless of the linker. Therefore, fluorescence is expected. The experimental run will ideally show fluorescence resulting from the self-excision of IN1 and IN2.

In the negative control (using the same constitutive promoter), GFP1 and GFP2, followed by a transcriptional terminator, flank RFC10 (Request For Comments) resulting in a stop-codon-containing scar. No fluorescence is expected for this component (background) because translation is interrupted. This is meant to control for the possibility of a non functional fusion protein. The expectation is that this fusion of GFP1 and GFP2 will not fluoresce, which is a consequence of some fusion protein techniques. Figure 6 shown below details the negative control design:

The figure below shows the construction map for the controls.

K576005 contains the first component of GFP (GFP1)

K576006 contains the second component of GFP (GFP2)

K576013 contains the promoter (P), ribosomal binding site (RBS), GFP and transcriptional terminator (TT). This is the positive control.

K576005 contains the first component of GFP (GFP1))

J61046 contains the lox site

K576006 contains the second component of GFP (GFP2)

contains the promoter (P), ribosomal binding site (RBS), GFP1, lox site, GFP2 and transcriptional terminator (TT). This is the negative control.

2.3 MAKING THE CONSTRUCT WITH RFC 53

1. 1) The insert is isolated through a series of enzyme digestions. One intron (in blue) is shown here as a representation. The insert is isolated for subsequent ligation.
2. 2) Similarly, the pSB1C3 vector is isolated through enzyme digestion. Note that "N" indicates that this is the vector portion. The vector is also isolated for the ligation step. It must also be noted that pSB1C3 vector contains a cut site of SacI, an enzyme that is used in RFC 53. Relocating the part in BBa_K371053 resolves this issue.
3. 3) The two components (insert and vector) are ligated together to produce the final construct.
4. 4) According to the experimental design, the final construct will contain self-excising ribozymes, which in the last step result in a non-disruptive ligation scar and, therefore, the expression of GFP.

2.3 Preliminary Testing

Although completion of a preliminary version of the final construct was achieved, lack of GFP fluorescence proved suspicion of questionable band placements during second and third stage electrophoresis. Final diagnostic digestion reaction confirmed abnormalities from designed constructs. Testing via digestion was completed for every intermediate, control and final constructs. Consequently, BBa_K576003, K576004, K576005, and K576006 were the only parts able to be confirmed. All the other intermediates and constructs have questionable band location which disrupted final construct fluorescence.

The above electrophoresis picture describes the resultant bands from the diagnostic digestion. Although bands 5, 6 and 7 (sub clones) have been confirmed, the adjacent positive control (band 8) and all GFP and intron digestions are not consistent with the expected patterns. The GFP-INT and GFP-INT-lox constructions (bands 9, 10 and 11) have been verified as inaccurate. The questionable placements of these bands indicate that the cut sites, thus the fragment length and containing sequence, do not match the planned construction. Therefore, it is not likely that they contain functional GFP, introns or lox, which would result in a lack of fluorescence in the final stage of construction. Further testing to reconstruct the contaminated clones is necessary for the functional final product; however, lab work has stopped due to time constraint. A diagnostic digestion at each step is recommended to circumvent any similar issues upon the continuation of this project.

3.0 PRACTICAL APPLICATION

The biggest advantage of the ribozyme project is the ability to create in vivo protein fusions. These can then be applied to a larger number of modular systems that can be used to make complicated expression systems. One such system is the creation synthetic antibodies. If protein sequences are flanked by intron sequences and then set up along the same stretch of DNA, different combinations of fusion proteins will be created based on how the intron excision occurs. Another system where the ribozyme project can be applied is DNA shuffling experiments. The Cry toxin is used as an effective biopesticide, however for now it has a very small range of insects that it effects. The ways to increase its range would be to change the structure of one of the vital domains so that it is able to recognize a wider spectrum of receptors in the host mid gut cell. To create different variations of this domains an in vivo DNA shuffling experiments using the ribozymes could be carried out.

4.0 REFERENCES

Belfort,M., Cech, T., Celander, D., Chandry, P., Heuer, T. (1991). Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core. Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado. 88(24): 11105–11109.

Belfort, M., Chu, F., Maley, F., Maley, G. and West, D. (1986). Characterization of the lntron in the Phage T4 Thymidylate Synthase Gene and Evidence for Its Self-Excision from the Primary Transcript. Wadsworth Center for Laboratories and Research. Vol. 45, X7-166.

Bernstein, K.E., Bunting, M., Capecchi, M.R., Greer, J.M., Thomas, K.R. (1999). Targeting genes for self-excision in the germ line.

Cassin, P., Gambier, R., Scheppler, J. (2000). Biotechnology Explorations: Applying the Fundamentals. Washington, DC: ASM Press.

Cech, T. (1990). Self-Splicing of Group I Introns. Biochemistry 59:543-8.

Clancy, S. (2008) RNA splicing: introns, exons and spliceosome. Nature Education 1(1). Genetics Primer, Fanconi Anemia Genetics. Last updated 08 February 2004. (http://members.cox.net/amgough/Fanconi-genetics-genetics-primer.htm).

Glick, B., Pasternak, J., Pattern, C. (2010). Molecular Biotechnology Principles and Applications of Recombinant DNA Fourth Edition. Washington, DC: ASM Press.

Goldberg, M., Hartwell, L., Hood, L., Reynolds, A., Silver, L., Veres, R. (2008). Genetics From Genes to Genomes Third Edition. New York: McGraw Hill Companies.

Group 1 Intron Sequence Structure and Database (http://www.rna.whu.edu.cn/gissd/alignment.html). Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado. 88(24): 11105–11109.

Ikawa, Y., Inoue, T., Ohuchi, S., Shiraishi, H. (2002). Modular engineering of Group I introns ribozyme. Graduate School of Biostudies, Kyoto University. 30(15): 3473-3480.

Landthaler, M. and Shub, D. (1999). Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: Introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes. Microbiology Vol. 96, pp.7005–7010.

Minnick, M.F., Raghavan, R. (2009). Group I Introns and Inteins: Disparate Origins but Convergent Parasitic Strategies. Journal of Bacteriology. 191 (20), 6193-6202.

Peters Ph.D., Pamela (N/A). Restriction Enzymes Background Paper An Excellence Classic Collection. (http://www.accessexcellence.org/AE/AEC/CC/restriction.php).

Self-Splicing RNAs (http://mol-biol4masters.masters.grkraj.org/html/RNA_Processing3C-Self_Splicing_RNAs.htm). http://www.bio.davidson.edu/courses/genomics/method/CreLoxP.html

1.0 INTRODUCTION

Since the Staphiscope project aims to detect S. aureus at low concentrations, it's important to determine how sensitive the system will be so that it can be adjusted to detect S. aureus at clinically-relevant concentrations. A detector that triggers at too low a concentration may display false positives, while one that triggers at too high a concentration may not give a positive when it should. To achieve the best sensitivity, numerical characterization for the Cambridge's 2009 sensitivity tuners needs to be obtained independent of the promoter used, in order to be combined with models for the AIP detection system and yield a predictive numerical model for Staphiscope. Work toward these ends is ongoing.

2.0 BACKGROUND

The sensitivity of Staphiscope may depend on various factors. For now, analysis has been restricted to just one factor: the choice of part used for the amplifier component of the system. The amplifier will be chosen from one of the 15 amplifiers submitted to the parts registry by Cambridge in 2009. Each amplifier responds uniquely to a given input signal, differing from the others with respect to its activation threshold (amoung a few other parameters less crucial to our analysis). Our goal is to determine which amplifier has an activation threshold in the correct range for the detection of S. aureus in relevant concentrations.

Empirical characterization of the response curves of each amplifier was carried out by Cambridge. However, in Cambridge's system the amplifiers were under control of the the pBAD promoter, which is not the case in Staphiscope. Therefore, the data gathered by Cambridge is not directly applicable to our system, since in general the response curve of each amplifier will be different under different promoters.

To obtain a numerical characterization of each amplifier, independent of promoter choice, we are undertaking the task of "reverse engineering" Cambridge's data to extract the parameters describing the amplifiers. A more detailed explanation of our approach first requires a description of the mathematical models relevant to this system.

3.0 SYSTEM MODEL

The parts characterized by Cambridge consist of a detector (the pBAD/AraC promoter) and one of the fifteen amplifiers. To obtain a mathematical description of the entire system, Cambridge used the following equations to describe the input/output response of these individual components.

The Cambridge 2009 modelling page develops these equations in more detail.

When these individual component models are strung together, the resulting model of the entire system has a sigmoidal shape. This means the response curve for the entire system can be fit to a Hill function, which is of the form .

Note that 4 parameters are required to specify the response curve: increase in rate, basal rate, switch point, and Hill coefficient. These parameters need to be determined empirically, with the exception of the Hill coefficient, which we assume is equal to 2.

The detector and amplifier also each have the form of a Hill function, but of course each has its own set of parameters which will differ from the Hill parameters of the overall system.

4.0 EXTRACTING DATA

Ultimately we seek a numerical characterization of the amplifier part alone. Since we know its response curve has the form of a Hill function, we need only find its four Hill parameters. The following table summarizes the known and desired data.

If all the parameters other than the amplifier parameters are known, the model above together with the response curves of the amplifiers is enough information to extract the desired parameters. Explicitly solving the equations above for the amplifier parameters is difficult; instead, MATLAB's curve fitting toolbox will be used to find the parameters which, when combined with the known parameters and inserted into the equations for Cambridge's overall system, matches the empirical data obtained by Cambridge. This process must be repeated for each of the 15 amplifiers.

Unfortunately, not all the necessary parameters are known. Some were not measured by Cambridge (those in block 1), but can be found in the literature. However, the Hill parameters for the pBAD/AraC promoter are not known, and must be measured. To do this, an experiment is being designed to characterize this promoter in RPU, but has not yet been carried out.

1.1 Outreach and Synthetic Biology

The newness of synthetic biology means that much of the population is not even aware that it exists (see Awareness & Attitudes Study see link at the bottom of this section). Therefore, an important aspect of our outreach efforts is to introduce the topic of synthetic biology and show its potential. We also hope to give people the foundational information that they need in order to understand future scientific developments. This form of outreach will help to improve the scientific literacy of the general population.

Another emerging issue is misconceptions held by the general public. In the development of synthetic biology, as with many new technologies, there is still much to learn and discover. As a result, the information made available to the public is often not a comprehensive, accurate picture of synthetic biology.

The following are the goals that we hope to achieve through educational outreach:

1.Inform the public about synthetic biology
2.Promote an education in science
3.Showcase opportunities in the field of science
4.Create an enriched science experience for students
5.Broaden the influence of iGEM

For more information about outreach and synthetic biology see:
This ground breaking study
by Peter D. Hart Research Associates, Inc. on awareness and attitudes of the public about synthetic biology found that 9 of 10 individuals think the public should know more about developing technologies

2020 Science
aims to provide a complete picture on the development of new sciences

Synthetic Biology Project
examines the development of synthetic biology

1.2 The Events

The method that we have chosen to achieve our goals is science education. This is the avenue most accessible and familiar to us as science students. By participating in events in our community we are able to influence multiple audiences by different means. Our outreach efforts are primarily focused at young students still deciding whether to continue in the field of science, students pursing science education, and the general public. Some of the events facilitated delivering the information on a small scale (in depth discussions with one or two students) while other events necessitated speaking more generally to larger groups. The events fostered synthetic biology awareness and were great learning experiences for our team.

ESQ Partnership

ESQ ( Engineering Science Quest ) is a day camp hosted at the University of Waterloo that brings hundreds of curious young minds to Waterloo each year to learn more about science and engineering. We held two different weekly activities for campers of ages 8-9 and 12-14.

The activity for children of 8 to 9 years of age involved extracting their own DNA from their cheek cells. It was approximately an hour and a half in length, and involved not only following the outlined procedure, but also, a discussion of synthetic biology, DNA, proteins, enzymes and more. At the end of each activity, the children were allowed to keep a sealed cryovial with their extracted DNA.

The protocol for the activity, as well as the PowerPoint presentation and script associated with it, are available here.

The second activity, which involved children 12 to 14 years of age was named “Do We Really Need to Wash Our Hands.” In this activity, the children were asked to swab their own hands (using a sterilized cotton swab) and plate the resulting swab on solid media. They would then wash their hands/use hand sanitizer and swab and plate again. The plates would be left to incubate at 37°C overnight, and the next day, the resulting growth would be presented to the children. As well, they were allowed to swab two other area of their choice, as to see how “dirty” these are. Most chose their own hair, backpack or shoe.

Prior to each activity, a PowerPoint presentation discussion bacteria, pathogenicity, hand-washing, DNA and synthetic biology was given to the students. As well, they were provided with a hand-out to aid them in their understanding of the activity. The handout contained step-by-step activity protocol, as well as spaces for hypothesizing and discussion. The PowerPoint presentation, protocol, script, and handout are available here.

We hope that these initiatives will help engage students outside of the classroom and get them excited about science.

iGEM Ontario (OGEM) Meeting, June 15 2010 at McMaster University

The second annual OGEM Meeting was held at McMaster University in Hamilton on June 15th, 2010. Members of iGEM teams from University of Waterloo, University of Western Ontario and University of Toronto gathered to discuss the future of a regional synthetic biology community, as well as a regional conference. The day turned over to discussions which centered around creating more communication and support between teams. In addition, the gathering was also an opportunity for teams to get to know one another before heading down to MIT. The meeting was a great success and the second of more regional gatherings to come.

As the meeting was held during the annual CSM (Canadian Society for Microbiologists) conference, members were able to attend a series of lectures given by valuable members of the field. As well, iGEM Ontario members were given the opportunity to speak about their projects and promote the idea of iGEM during a poster presentation held along with researchers in the field of microbiology.

In the future we hope to see this organization (independent of an individual iGEM team) become an important resource to Ontario iGEM teams and for educating the general public about synthetic biology. We are currently working on an iGEM Ontario website.

Building Life: The Science of Synthetic Biology

On June 23, Waterloo iGEM adviser, Dr. Trevor Charles, held an open public lecture aiming at discussing synthetic biology, its means and aims. During the lecture, the purpose of synthetic biology, its ethical and safety implications well as many other current topics were discussed. At the end, a facilitated panel discussion featuring Andre Masella (Waterloo iGEM team), Dr. Kathryn Plaisance (bio-ethicist), as well as Dr. Maria Trainer (Council of Canadian Academics) answered some of the public’s pending questions regarding synthetic biology. The lecture was a great success, with a large number of attendees of various scientific background. It was part of a series of lectures organized by the University of Waterloo’s Department of Biology, in attempt to increase public awareness of current scientific issues.

What We Accomplished

Upon reflection we feel that we have achieved the following goals through our outreach efforts:

1.Introduced synthetic biology to individuals who knew nothing about it
2.Educated multiple audiences on the fundamentals of science such that they will be able to better understand future scientific information and developments
3.Excited young minds about science
4.Strengthened our regional synthetic biology community
5.Communicated the work of other iGEM teams and the benefit and goals of the iGEM competition
6.Increased communication between local synthetic biologists
7.Gained insight and experience into what the public believes about modern science
8.Developed activities and displays to be used and improved upon in the future

Future Plans

We plan to continue our outreach efforts in the future. We plan to expand our efforts and continue to educate the public about synthetic biology and share our love of science. In the future our efforts will continue to center around science education. Our profile in our community is increasing as iGEM becomes a familiar group and synthetic biology ceases to sound frightening and gains familiarity. We hope that the Ontario community of iGEM teams will continue to grow and include teams from all across Canada, helping to strengthen the synthetic biology community across the country. As members of the Waterloo iGEM team we are proud to educate multiple audiences and to share our knowledge and passion with the world.

INTRODUCTION

As synthetic biology expands, as new innovations are made, and pressing world problems are solved, the potential impact synthetic biology will have on the world becomes more evident. Although a primary goal of developers of synthetic biology should be to consider the ethical, societal, safety, environmental, and political impact of the science, we believe that interest should also be paid unto the impact that synthetic biology will have in the business world.

As a group we are most interested in the course of development of synthetic biology in industry; the goal of our project is to try and decipher the path that synthetic biology will forge as it expands in the business world.

Our attempt to answer this question has begun with a comprehensive inquiry into important factors affecting the diffusion of synthetic biology. It is our opinion that before we attempt to make any conclusive statements about the future direction of synthetic biology or the economy as a whole we must have a complete picture of the current landscape of synthetic biology and the markets it could potentially impact.

This inquiry is intended for use by multiple audiences; particularly scientists and members of business in relevant industries. As scientists it is important to understand the context in which discoveries are made, understanding where world needs lie and how discoveries will impact the world makes for better-informed scientists. Members of the business world should also strive to have an understanding of where the need and rationale for discoveries come from. Although profit is the ultimate endgame a well-informed approach helps to prevent ethical pitfalls and often greater success.

This analysis looks at both extrinsic and intrinsic factors relating to the development of synthetic biology as a whole. In particular intrinsic factors such as the development of synthetic biology in specific industries (biofuels, pharmaceuticals, and bioremediation) is examined in depth. Important extrinsic factors such as the impact of patenting and open source are also analyzed. With this information we feel we have laid the foundations for a comprehensive inquiry that will allow us to better understand what the expansion of synthetic biology in the business world will resemble.

OPEN SOURCING and Synthetic Biology

What is Open Sourcing?

What differentiates synthetic biology from biotechnology is that it offers the creation of systems or pathways that would not be found naturally in an organism. Synthetic biologists believe in a standardized system of parts similar to that of electrical engineers having standard circuits and components¹. This is also similar to how LINUX modules have been combined to create different software. This contrasts the closed-parts strategy where developers use such methods as patent protection and secrecy to gain a competitive advantage over others². That is why the Massachusetts Institute of Technology (MIT) has created the initiative for what is known as the “Registry of Standard Biological Parts” where it indexes biological parts that are currently being built. A standard array of modular gene switches or parts that can be found from a common library and can be mixed and matched in various combinations, which is the goal that synthetic biology targets. This is a similar path as what happened twenty years ago when software became standardized and allowed Microsoft to become a monopoly. The issue in the air currently is if this could happen with the synthetic biology industry³.

Advantages

Open-sourcing has been an idea that has been the basic foundation of synthetic biology throughout the years. Supporters strongly believe in a world where companies and academia will be able to develop and share parts freely for the advancement of the whole field, not just a singular firm or university⁴.

A term that is used commonly in this industry is called network effects. This means that the more a product is used, the more attractive it becomes. Over time as each part is used repeatedly on a specific metabolic pathway, especially when in successive experiments, its cost goes down. With the limited data that is available, it is predicted that total project costs could be cut down by 25% after its first successive use. It is also likely that these costs would be cut down several more times until it flattens as with each subsequent experiment more knowledge on that part is gained and intuitively, less errors are made.

One such example is with Amyris’ artemisinin project that spanned over the length of five years, costing $20 million. It was reported that 95% of the time spent was on finding and fixing unintended interactions between parts.

Due to the fact that one part must be used in conjunction with other sets of parts gives incentive to companies to create whole libraries. This is very similar to how software companies develop several programs that are able to cover multiple applications. Not only that, but there is opportunity for these companies to make profit by patenting some of these parts and making others openly available due to the strong modularity of the open-parts approach.

It is expected that companies will have their own individual parts needs. This means that other companies cannot just sit around and wait for another to develop a part. This also gives a type of competitive advantage as companies that share parts will not be losing their, ‘technological edge’ to other competitors. Since different companies have idiosyncratic needs and hence expertise, it proposes that community-based libraries will outperform individual companies. The industry will probably have a large number of small, distinctive customers, meaning that patent licensing will be less attractive and the open-parts initiative more so⁵.

Normally, the value of a patent depends on the inventor’s initial R&D investment, but with synthetic biology parts it would depend on that as well as how many researchers have subsequently used that part. According to the law, this allows patent owners to capture both sources of value, which can be unfair to society as they must deal with high prices without getting anything more in return. The open-parts initiative sets the price of parts equal to zero, so it is naturally able to solve that problem. There are two incentives that will draw companies to an open-parts initiative. The first would be the opportunity to share R&D investments among multiple firms and secondly the opportunity to produce parts faster due to shared insights⁶.

Barriers and Disadvantages

About twenty years ago when software became standardized, it opened the path for Microsoft to come in, take over the industry and monopolize it. Can something similar potentially happen in the synthetic biology industry? At the moment, synthetic biology is what is called a ‘tipping market’. It is unstable and prone to monopoly. Building on this, the tipping dynamic is indifferent to whether or not the dominant parts constellation is open or closed. If for instance a mature industry is able to grab a hold of these shared parts, it can work towards this open-parts initiative. Though on the other hand, companies, out of necessity will also pay for closed parts, both solutions are equally viable.

Some other issues include an agreement on standard nomenclature for the parts, therefore when actually designing a database, controversies might come up. As well when collaboration occurs there must be adequate legal infrastructure. This must include a license specifying the rights and duties of members. One of the main legal problems is that gene data is unlike software, it cannot be copyrighted.

Many pessimists towards the idea say that it is now too late for synthetic biology to use an open-parts collaboration. Now with Amyris’ advancement in the field it seems that this industry might monopolize and follow the path that Microsoft had. The fact that commercial synthetic biology receives so much government support shows a bit of laissez faire attitude. In America’s Department of Energy $350 million biofuel initiative there was no open-parts requirement at all. It is said that at least when Bill Gates cornered the software market he did it with private money⁷.

¹ http://heinonline.org/HOL/LandingPage?collection=journals&handle=hein.journals/tlr85&div=50&id=&page
^2,3,4,6,7 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726678/
⁵ http://www.nature.com/msb/journal/v3/n1/full/msb4100161.html

PATENTS and Synthetic Biology

The ideological reasoning behind applying for a patent lies in the fundamental incentive that it would bring forth financial rewards and a sense of monopolistic supremacy in an advancing market. The imposition of patenting technology has been deemed useful in many industries, most notably in electronics. However, it has also brought forth a considerable level of controversial dialogue in determining how historical protocols can be implemented within new technological streams that sometimes question the concept of man-made invention. For example, Craig Venter’s desire in protecting his synthetic cell research methodology gives him a deserving ownership, but his broad patent places a downgrading threat towards the field of synthetic biology. Ironically speaking, patenting is a legal practice that promotes innovation, but imposes a sense of apprehension for those who wish to adapt towards a more open-sourced strategy. This analysis focuses on the implications of introducing patent law to the field of synthetic biology as well as the practicality of gaining rights to a product not developed entirely by man, but with elements arising from nature.

Monopolizing Synthetic Biology

The accrued financial and proprietary benefit of monopolizing an idea within a competitive market is indeed an accelerated advantage when a patent is granted. However, many have the misconception that monopolistic proprietorship has a standardized value to any industry it is applied to. For years, Professor John Sulston from the University of Manchester, a believer in promoting an open-source environment in the field of synthetic biology has proposed the implications that would arise should Venter receive approval upon his protocol in developing synthetic organisms. Sulston states,

"I hope very much these patents won't be accepted because they would bring
genetic engineering under the control of the J Craig Venter Institute (JCVI). They would
have a monopoly on a whole range of techniques (BBC UK, 2010)."

The irony in this matter is that the technological industry used the concept of monopolistic competition to drive both innovation and the development of products that would eventually overpower the market leaders. The reason that this concept cannot presently be applied is that synthetic biology is strongly reliant on collaborative construction. Patenting biological parts or processes will not motivate a group of scientists to pursue their research, but cause them to weigh the opportunity costs between their groundbreaking research and the hefty payments they would make to patent holders.

In the case of Venter’s claim, organizations such as the ETC group believe that Venter’s group is eyeing on a profit making opportunity. Hope Shands of the ETC group states, “The fledgling synthetic biology industry keeps talking about how they’re going to fix climate change – but these sweeping patent claims reveal that the companies are much more focused on securing profits than on human needs” (ETC Group, 2007). The multi-purpose use of a biological methodology would not only give Venter ownership to a scientific methodology, but to a range of applications that could be possible in the chemical, medicinal or environmental industries. Venter reported to Business Week, “If we made an organism that produced fuel that could be the first billion- or trillion-dollar organism. We would definitely patent that whole process” (ETC Group,2007). Many would dispute that this financial and market acquisition would only de-motivate other synthetic biology scientists and facilities to halt their research. But from looking at it from Venter’s perspective, wouldn’t anyone want to reap the rewards on a project that took 15 years and 40 million dollars to reach its success?

Patenting Artificial Life: Is synthetic biology a man-made industry?

As previously noted, synthetic biology encapsulates the field of biotechnology, software and computing. The current patent laws that have been put in place have generally been applicable to all industries individually. However, the field of synthetic biology is an amalgamation of two fields that have faced years of controversial debate in terms of granting or approving patents. The concern lied in the fact that patents related to inventions in biotechnology or software could be broad or narrow, but extensive enough to “hold-up” the concept of innovation and invention (Rai and Boyle, 2007 ).

Synthetic biology is being renowned as a stream towards the development of artificial life, which for some raises social, ethical and legal concerns. Patent Act 101 states that any subject matter that is found in nature is not deemed as patentable. However, if the product found in nature is modified or transformed to something that is novel and non-obvious, it holds credibility in attaining a patent. Looking at the synthetic cell created by Venter’s team, it is difficult to denote the “man-made” material in his composition. His cell includes a computer generated “minimal genome sequence” that is encapsulated within a bacterial cell (found in nature) and still hosts cellular machinery required to allow the cell to adapt and function within its environment. The modification is within the genome, but even that raises some debate on whether it can be patentable.

The software industry has always tackled with patenting mathematical and computational algorithms, particularly in concern was their level of broadness. In perspective to the field of synthetic biology, an algorithm is analogous to the creation of novel and modified genetic sequences, which give rise to new metabolic pathways and cellular functionalities. Yet again, the derivation of their new genetic sequence comes from the naturally degenerate genetic code, bringing up the question of whether this is entirely man-made. What is seen here is a disconnect, in that it would be difficult to set fundamental patenting protocols for the field of synthetic biology, given the legal complications faced in both the field of biology and computing. This is where many may agree that an open-source strategy such as the MIT Registry of Standard Biological Parts would bring forth more progression, versus the time and effort it would take to validate the patentability of a biological part or process.

SWOT Analysis

Below are other indicators to help define the relative benefit of introducing patent law, as well as the long-term threats that may hinder the advancement of research in the field of synthetic biology.

Strengths

1.Patenting assists in stimulating investment, and secured investments bring forth progression in research. Given that research projects can cost up to hundreds of millions of dollars, patenting synthetic biology technologies would provide a more steady approach in financing the development of an invention.

2.Developers are motivated by reward, which is provided by the successful implementation of patent law

Weaknesses

1.The field of synthetic biology is different in the sense that innovation is not promoted through attaining proprietary rights to an entire process or genome, but through the collaborative use of fine, functional and specific biological parts

2.Synthetic biology is multidisciplinary as it incorporates the field of biotechnology and computing, each has their own sets of legal rights, restrictions, and pitfalls

3.In addition, lawyers would require a more extensive level of knowledge and proficiency in both fields

4.Sometimes, hundreds of parts are necessary for the composition of one biological machine, and attaining rights to each part would create what is called to be a "patent thicket" (Rutz, 2009). Attaining rights to so many parts does not only hinder innovation, but is time-consuming and costly

Opportunities

1.Patents add value, reputation and credibility to an invention. Although this legal concept is frowned upon in the R&D field, it brings a source of financial opportunity that would bring forth strategic partnerships and lump some funding to further progress research. At an economic perspective, patenting would be useful in advancing one's research.

2.Patenting requires thorough documentation and characterization of each aspect of the part, streamlined patenting protocols could add standardization to the part development process

Threats

1.European patent law states that "iventions of commercial exploitation to morality are omitted from patentable rights" (Rutz, 2009). The general public fears that if rights to a biological process or part are give they may be abused. Examples are biological warfare or the unintentional release of pathogenic organisms.

2."Patent sharks and trolls" (Rutz , 2009) may find it simple to file lawsuits against such patents. •On the other side of the coin, some have claimed that finding a patent application for a biological part is similar to finding a needle in a haystack. Moreover, it is not the job of a scientist to be rummaging around in identifying his legal rights to using a biological part, nor should his time be consumed by understanding the patenting process relevant to his field.

Conclusion

The concept of patenting makes scientists cringe at the thought that their research and advancements would be diminished by the monopolistic power held by the broad patent holders within synthetic biology. There is currently no established set of specific protocols for patenting products in synthetic biology. Moreover, the attempt to blend the patent laws applicable to a range of disciplines to one as intricate as synthetic biology is difficult. Additionally, companies who wish to have a formalized process in examining and submitting processes would have to understand that a large number of additional resources would be needed to accommodate for the labour intensive, time consuming and costly process of patenting such biological parts. So the underlying question is that what is of greater value in today’s market, leading innovation through open-source strategies or banking on profit-making opportunities by securing an idea with a patent?

INDUSTRY ANALYSIS

Before we attempt to understand the impact of synthetic biology on the business world as a whole, we must first understand what kind of an impact it will have on specific sectors. The goal of the industry analysis was to pick specific industries that we felt would be heavily impacted by the emergence of synthetic biology and attempted to understand their current situation as well as opportunities and threats facing each industry.

Biofuel Industry

The oil industry has become an integral part of the society we live in for, transportation, food, healthcare, and communication. However, there is a dire need for alternative sources of energy and the world is beginning to turn towards biofuels for support in this area. Although there is immense promise in this area there is also many challenges to overcome such as reliance on environmental factors and adequate feedstocks. Although emergence of biofuels created through synthetic biology does not have the market potential to shift the whole method of biofuel production, politicians are optimistic that it has the potential to make an impact and have therefore taken measures to support the infrastructure of its’ development. The emergence of a viable synthetic biology biofuel would serve to excel the development of synthetic biology, particularly on the business stage.

Pharmaceutical Industry

Our knowledge of diseases and treatments has advanced to an exciting point as has society’s perception of health problems and issues. However, as fast as our knowledge advances, diseases like H1N1 and HIV provide a pressure for the pharmaceutical industry to advance further and faster. In addition, aging populations and growing urban centres poise pharmaceutical companies for significant and meaningful innovation over the coming decades. The pharmaceutical industry is expected to grow to an $800 billion industry by 2011, expanding as a global force. Thus, synthetic biology, although faced with challenges in terms of legal and social concerns, is poised to have a significant impact on the pharmaceutical industry. Not only is there promise for curative treatments that would change how we view illnesses like HIV, there promises to be significant implications in the social, technological, and political realms. Unanswered questions about the handling of intellectual property issues will challenge the development of synthetic biology in the pharmaceutical industry as will ethical, safety, and political issues.

Bio-remediation Industry

Unlike other industries, bio-remediation is an industry that has incorporated the use of genetic engineering and synthetic biology since the 1970’s. Based on the research conducted, there is a definitive understanding that this field poses an optimistic approach in achieving results that are environmentally friendly and cost-effective. The major setback with the field of bio-remediation is the intricacy of the techniques that are involved. Aside from the general publics qualms about bio-remediation procedures, even the scientific community does not have a holistic understanding of how the processes within bio-remediation work, or how to optimize a microbe’s “oil-eating” activity based on its metabolic characteristics. Most say that bio-remediation is a cost effective technique in treating vast oil spills, but it seems that investors and economists have not incorporated the cost of time and labour required in mastering the techniques involved. Once these techniques have established, there is a definite opportunity for this field to overpower the current invasive chemical processes that are being used as a last desperate resort in treating these environmental disasters.

The project wiki contains a condensed overview of the industry analyses, if you are interested in learning more be sure to check out the complete industry analysis.

Conclusions and Outlook

After conducting this research into the current and future position of synthetic biology particularly in the context of the business world, we are optimistic about the course that it will take. There are significant hurdles to overcome but overall there is a sense that synthetic biology will impart a positive impact not only in terms of specific products but also in terms of micro and macroeconomic impact.

In this inquiry we have attempted to understand some of the important issues facing synthetic biology from a business perspective. In the future we hope to take our work a step further; we hope to take our knowledge, thoughts, and questions into the business world. Through interaction with business owners, scientists, legal specialists, and other stakeholders we hope to disseminate our findings and build on them. In the future we will continue to focus on our goal of deciphering the path that synthetic biology will take as it emerges in the business world.

References

1)Rai A, Boyle J (2007) Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons. PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058

2)Barrett, Margaret. Intellectual Property. 2nd ed. New York: Aspen Online, 2008. 33-34

3)Dickinson, Boonsri. "Will Patents Give Craig Venter a Monopoly over Synthetic Life? - SmartPlanet." SmartPlanet - We Make You Smarter - People, Business & Technology. 28 May 2010. Web. 26 Oct. 2010. .

4)"Artificial Life: Patent Pending | The Economist." The Economist - World News, Politics, Economics, Business & Finance. 14 June 2007. Web. 26 Oct. 2010. .

5)"Postnote: Synthetic Biology." Parliamentary Office of Science and Technology, Jan. 2008. Web. .

6)Rutz, Berthold. "Synthetic Biology and Patents : A European Perspective." Nature Publishing Group : Science Journals, Jobs, and Information. 2009. Web. 26 Oct. 2010. .

7)Joachim, Henkel, and Maurer Stephen. "The Economics of Synthetic Biology." Molecular Systems Biology. Nature Publishing Group, 5 June 2007. Web. 26 Oct. 2010. .

8)Ghosh, Pallab. "BBC News - Synthetic Life Patents 'damaging'" BBC - Homepage. 24 May 2010. Web. 26 Oct. 2010. .

9)Ball, Philip. "Biology News: The Patent Threat to Designer Biology." BioEd Online. 22 June 2007. Web. 26 Oct. 2010. .

10)ETC Group. "Extreme Monopoly: Venter's Team Makes Vast Patent Grab on Synthetic Genomes | ETC Group." 8 Dec. 2007. Web. 26 Oct. 2010. .

11)Hammond, John, and Robert Gunderman. "The Limited Monopoly- Patent Law 101: What Is Patentable?" The Rochester Journal, June-July 2007. Web. 25 Oct. 2010. .

UNIVERSITY OF WATERLOO

University of Waterloo was founded in 1957 and has grown to accommodate 30,000 undergraduate and graduate students, and has become Canada’s leading university in comprehensive learning. Also, the university has consistently been voted as the most innovative, most likely to produce the leaders of tomorrow, and best overall University in Canada for over 18 years (according to Maclean’s Magazine). Waterloo’s reputation is however based on its excellent and pioneering co-op program which offers students a balance of work and school on a per term basis, making it a unique learning experience. The city of Waterloo has recognized University of Waterloo and its students, by meeting its demands in terms of funding and involvement. The University has also opened up two new campuses; the pharmacy building, and the joint McMaster medical building in Kitchener, as well as the architecture building in Cambridge, contributing to not only the city of waterloo but the whole Grand River area.

WATERLOO - KITCHENER COMMUNITY

City of Waterloo mainly revolves around the two universities: University of Waterloo and Laurier University. Waterloo is surrounded by Kitchener and thus, the two cities are known as the twin cities, also referred to as Kitchener - Waterloo. The population of the city of Waterloo is always fluctuating due to temporary residents at Waterloo’s two universities. Total population in 2009 was recorded to be 121, 700; approximately 20,000 of which were temporary post-secondary students. Due to its small size, people in the past have tried to merge the two cities together but have been unsuccessful. As of today, both cities have their own identity and their own separate city governments.

Lab Notebook 2010

Monday, May 10

We're still working on 2009 project. We just found out that the donor strain sequence lacks att site and oriT. Inoculated liquid broth in order to obtain colonies for conjugation. The tubes were as follows:

1.DH5α λ rifr attB CT from old patch on rif25, amp100, tet10 (aka RAT)
2.DH5α rifr attB CT from old patch on RAT

Also, we inoculated the Landing Pad Strain cells into liquid broth.

Tuesday, May 11

Tubes incubated from yesterday were taken out. All showed no growth. All tubes are to be re-innoculated. Tubes #1 and # 2 were re-done using an old plate and different colonies of the old patch.

Several dilutions were prepared in order to determine which concentration is best for spread-plating. Dilutions prepared were as follows: e-2, e-4, e-5, e-6, e-7, e-8

Prepared liquid and solid media, and learned how to use autoclave.

Made more plates (Rif/Amp/Tet/Sm)

Wednesday, May 12

All tubes incubated yesterday showed growth. Miniprepped the samples.

As relating to the dilutions, results were as follows

e-8 = very little growth e-7 = some growth e-6 = highly populated

Conjugation experiment was attempted (tri-parental mating). This included Donor (DH5alpha), Recipient (MM294A), Helper(MT616)

Inoculated Landing Pad Strain from frozen stock.

Thursday, May 13

We prepared X-alpha-gal plates and streaked MM294A on them. We would be expecting white.rosy colonies. Nanodropped the samples which were minirepped yesterday (incubations of plates listed on Monday, May 10.)

Friday, May 14

The x-alpha-gal plates showed no good results - none of the colonies were rosy.

Learned how to do REs digestions properly (did several examples on paper, and in the lab.)

Monday, May 17

Planned how to do a restriction digest followed by ligation procedure for the donor strain. Discussed the different purposes for restriction digests: diagnostic (to check if you have the plasmid you expect) and for cloning (to isolate desired DNA).

Tuesday, May 18

LPS Analysis W.R.T SacB : LPS was cut with with EcorI & run on 0.8% agarose gel for 45 minutes at 90V to analyze for truncated sacB

Results: Three distinct bands were found. Therefore, the truncated sacB was not present in the LPS. If sacB were present four bands would have appeared after digestion.

Wednesday, May 19

PCR of DS Right Plank was performed: The right flank was inserted into DS right flank=Pst site, attB, oriT, MPH 1103

PFB 9009 was mini prepped(LPS) and nanodropped.

Nanodrop results of PFB 9009 Mini - prep:

SAMPLE------------>Concentration(ng/µL)----->260/280
PFB9009-1--------->21.4.....................>1.75
PFB9009-2--------->24.0.....................>1.91
PFB9009-3--------->12.9.....................>1.58

Thursday, May 20

Performed a double digest of PFB 9009-2 with FSPI & NdeI to excise and extract rouge transposase gene. Gel extracted the LPS and DS fragements LPS: 0.125g DS: 0.0947g

Dissolved in Buffer to obtain 500µL of LPS and 378µL of DS and extracted the DNA following the protocol from "forEZ-10" kit.

Friday, May 21

Performed a ligation reaction of LPS to circularize it Nanodropped the samples. Results recorded below:

LPS Concentration: 2.06ng/µL 260/280: 5.6 DNA: 224ng
DS Concentration: 1.95ng/µL 260/280: 7.1 DNA: 284ng

The solutions were speed-vaced on high for 10 mins to increase concentration.

Monday, May 24

Prepared agar plates and agar bottles

organized everything in the lab and the freezer so that it was easily accessible

Construction tree was revised and updated

Tuesday, May 25

Planned and carried out ligation of DS right flank + DS2(blunt ends) Started transformation of LPS and DS Note: The desktop cooler has been left out for undefined amount of time (approx. 3 hours). However, it should be okay.

Wednesday, May 26

Transformation results: LPS did not grow. Therefore, reverted to double digest.

Started double digest of LPS with PspI and NdeI

Repeated transformation (using more DNA this time)

Thursday, May 27

Transformation results (pFB10, Km + Sm):

negative control: no growth
Positive control: lots of small colonies (approx. 70)
Therefore transformation was sucessful.

Friday, May 28

Performed a diagnostic digest on DS with PstI + NsPI
Results: Failed. NO 274 fragment was present but 120 fragment was present.

This will be attempted again.
Troubleshooting: Ladder will be added in higher concentration and no loading dye will be added for better resolution.
Integrase strain was inoculated into Tc10 LB broth

Monday, May 31

Miniprepped and nanodropped DS
Results:

SAMPLE 1
CONENTRATION: 110.14ng/µL
260/280: 2.02

SAMPLE 2
CONENTRATION:127.85µL
260/280: 1.06

SAMPLE 3
CONENTRATION:105.89ng/µL
260/280: 1.07

SAMPLE 4
CONENTRATION:189.74ng/µL
260/280: 2.03

SAMPLE 5
CONENTRATION:152.01ng/µL
260/280: 1.05

Planned out next day's activities regarding DS

Tuesday, June 1

Retried to do a diagnostic gel of DS with PstI & NstI with a fragment known to have approx. 100bp(old DS with no right flank) FAILED. No alkaline phosphotase was added to DS when digested with restriction enzymes
Started a RE digest of mini-prep DS plasmid to be re-ligated with right flank construct. Nanodropped the digested right flank sample.

Concentration: 7.8ng/µL
260/280: 4.29

The concentration indicates that we can not use this sample because majority is not DNA.
Therefore, must PCR the right flank again using the "spring 2010" PCR program
PCR products were digested with Mph1103 & PstI
DS was digested with right flank with Mph1103(AvaIII) & PstI to prep for insertion into DS plasmid.
Two bands were present for positive controls#2(both samples). These bands were extracted and purified.
TUBE WEIGHTS

Sample 1:0.1633g
Sample 2:1.0477g (did not show bands).

NANODROP RESULTS

Insert

Concentration: 15.9ng/µL
260/280: 1.61

Vector

Concentration: 8.4
260/280: 2.23

Wednesday, June 2

Digested DS vector with PstI so that right flank can be inserted. Added SAP to avoid re-ligation with itself.
Ligated DS vector with Right flank insert
Transformed newly improved DS(hoepfully, with right flank) into component DH5α
Discussed possible ligation results

Thursday, June 3

Innoculated S. aureus into LB media for the purpose of collecting AIP sipernatant
After collecting staph supernatant wanted to test for the effect of adding supernatant to E.coli cultures

Friday, June 4

Innoculated DH5α into AIP supernatant
made 3 x 5mL tubes of AIP supernatant
innoculated with DH5α (strain box #1, #41)
Results from transformation(previous day): No growth of LPS 10 on Km/Sm plates. No clear red color on Rig/Km but good growth observed. Left in the 37˚C incubator over the weekend.

Monday. June 7

Performed another attempt at digestion of diagnostic Nsp/Pst on DS -- FAILED
Researched and wrote protocols for experiments to be carried out within the week
Learned how to use DNA from kit

Tuesday, June 8

A new batch of competent cells were made.
Their competency will be tested by transforming DS in.

Wednesday, June 9

The streaks of putative LPS 10 still showed no red.
Sensetivity was checked for melibrose. The same patches were streaked(#5,8,26) on minimal melibrose.

If anything grows: ALARM! Also, there are suspicious parts on #5 streak, worth restreaking onto another.

Thursday, June 10

Rif/Km plates were made
Rif stock was made by dissolving 0.25g into 10mL DM50
Prepared everything needed for outreach event
pJET-Right Flank for DS (Donor Strain) has been miniprepped, put in -20C freezer. The streak plates of LPS10 from various sources still show no red colour. Could cutting out the fragment (pFB09->pFB10) messed something up?

Friday, June 11

Tried to cut and gel extract the fragment. Failed, probably due to low Gel Red dye. Will be getting more dye soon. Can try scraping the walls and adding all of it to the gel Monday.

Monday, June 14

Tried to do PstI + Mph1103I digestion again. Failed several times.
Wednesday will do 10uL diagnostics with BglII on the remaining pJET minipreps; looking for ~200bp fragment. Also will do spectrophotometry of DH5-alpha pre-DS and DH5-alpha blank hourly at 600 and 640 nm.

Tuesday, June 15

Updated construction tree. Drew one out and many copies were made.
Attended a club meeting and updated all volunteers with what was happening.
Engineers were given a short biology course to help understand the project better

Wednesday, June 16

Checked concept of absorption RFP estimation
inoculated with loop (approximate the same aount) HiRFP = pSB1A2 - BBa_K093012 (+ L. flank, can be neglected)(J23118 driven E1010)
The purpose of this was to check for consistency of A589/A640 on DH5α and useage of that to check consistency of A584-blank / A640

Thursday, June 17

Gel extracted the fresh right flank PCR product (yes, AGAIN). Tomorrow will check on gel for presence along with "control PCR product" from cloneJET kit.

The parts in DH5 have been inoculated into liquid media to be miniprepped tomorrow.

The RFP measurements on cuvete absorption spec Ultrospec 2000 suggest that sensitivity is too low to pick out RFP.
Should try on Bioscreen C plate reader.

Planning the assembly should be started tomorrow.

Friday, June 18

Parts miniprepped. right flank PCR ligated into pJET and transformed. To be inoculated to liquid Monday.

Monday, June 21

created a final list of parts to be transformed
Do not have: K206000, K206001, J23151 and J23150
All the DNA was transformed into DH5α competent cells
Results of transformation: most plates grew but with very few small colonies. The plates were put back in the incubator for a few hours and checked later. All had at least a few colonies.
These were innoculated into liquid LB and incubated overnight at 37˚C

Tuesday, June 22

Was Done:

Tried diagnostic with Mph1103I alone and PstI alone. The results are here. Wells are as follow
1-2 PstI
2-1 PstI
3-1 PstI
Ladder Fermentas 1kb plus
1-2 Mph1103I
2-1 Mph1103I
3-1 Mph1103I

The evidence hints on presence of PstI site on pJET; without first well showing it is quite non-conclusive.
Red clone of LPS10 strain was found in the fridge and confirmed by X-gal to be MM294A as DH3-alpha is lacZ-. Hooray.
Was patched on Kan and Kan+Strept 48-numbered background plates by Corey and Diana.

To Be Done:

Talk to people who use Bioscreen C plate reader.
See if pJET sequence has PstI site(s).
Cut out right flank with PstI and Mph1103I and run gel preparatively, gel extract the fragment of ~150bp.
Ligate that fragment into pDS (donor strain plasmid) cut with PstI+SAP.
Transform that ligation into DH5-alpha.
From LPS10 patch plates pick the colonies that did not grow on strept or grew poorly, streak on Kan and check on sucrose media (just streak) with empty DH5-alpha as control.

Wednesday, June 23

Was done:

Looked at pJET for PstI. Yes there is one, at position 5. This means that in Mph1103I+PstI digestion we expect fragments of of 153bp (the one we want) and 371bp (the one we don't care about).
Spread 70 µL of red LPS10 on X-alpha-gal+rif+kan.
Streaked red LPS10 on sucrose LB side by side with empty DH5-alpha
no streptomycin sensitive patches detected
Mph1103I PstI digestion preparative was done, gel was stained overnight.
pDS was linearized with PstI

Thursday, June 24

LPS10: sucrose sensitivity not detected; X-alpha-gal plate is mostly blue, only few white colonies, without a sign of red. Should leave x plate in the fridge and hope for colour
Two attempts to cut out right flank with Mph1103I and PstI were perfmored. Both attempts are to be quantified using nanodrop and pipette, and concentrated on the Speedvac, requantified with pipette.

Friday, June 25

Second attempt of Mph-Pst cut out has been ligated into its proper place in pDS. incubate and pray. Tschüs.

Monday, June 28

PstI+SpeI digest (wells 4 and 5) suggest ligation failure of right flank into pDS.

Tuesday, June 29

Transformed J23107 (constitutive promoter) into DH5α
Results: colonies grew, some rosie
These were inoculated and miniprepped
All parts were sent out for sequencing

Wednesday, June 30

Digested RF with PstI.
Result: No RF fragment, but PstI- PstI fragment is there

Thursday, July 1

Attended board meeting and updated everyone on what was going on
Made LB plates

Friday, July 2

Autoclaved 0.8% saline and swabs
make 70% ethanol
stocked up pipettes

Monday, July 5

Inoculated J23107 in LB and incubated overnight in 37C
Inoculated received parts( K206001, I746201, I746001, K20600 onto Amp plates to incubate overnight in the 37C. It will be inoculated in LB tommorow

Tuesday, July 6

inoculated the cultures into LB and will mini-prep tommorow

Wednesday, July 7

Mini-prepped the parts that were inoculated yesterday
These were to be sequenced
Made glycerol stock of parts to be sequenced

Thursday, July 8

pIET - rf has been transformed and grew up to nearly lawn coverage o/N. 50uL DH5a comp + 5uL supercoiled plasmid were used. The negative control was clear. The transformants were inoculated into 16 LB tubes + Amp, without purification.
attempted to assemble the measkit.

Friday, July 9

Nanodropped the following samples:
J23107 - COnstitutive promoter
I74601 - AIP sensor
746001 - AIP generator
I746201 - FepA
I7466104 - AgrA P
K206000 - pBAD strong
K2066001 - pBAD weak
I13458 - pBAD
I13453 - AraC
J23102 - constitutive promoter
E1010 - RFP CDS
J23101 - Constitutive promoter
I13507 - RFP + RBS +TT

Monday, July 12

Made frozen stock of all the cultures from friday
Made DMSO stocks : wanted 7% DMSO and 14% LB --> Made it by mixing 1.68mL of DMSO and 10.32LB

Wednesday, July 13

miniprepped all parts from strain box #2 and nanodropped them. Results are listed below:
I746104 : 100.1ng/uL , 2.16
J23107 : 149.3ng/uL , 2.01
I746201 : 139.7ng/uL , 2.00
I746001 : 185.9ng/uL , 1.96
I746101 : 104.3ng/uL , 2.08
J23102 : 319.4ng/uL , 1.94
I13453 : 139.2ng/uL , 2.01
I13507: 61.0ng/uL , 2.10
K0206000 : 155.1ng/uL , 1.60
I13958 : 155.7ng/uL , 2.03
J23101 : 262.8ng/uL , 2.00
E1010 : 99.1ng/uL , 2.05

Thursday, July 14

Added J23107 promoter to I746101 and I746001 (AIP sensor and AIP generator)
Ran gel electrophesis and gel extracted J23107 ( approx. 3kB fragment)
Cut J23107 with SpeI and PstI & I746101 and I746001 with XbaI and PstI and ligated into vector
Transformed and miniprepped.

Friday, July 15

Strain list made public with the link
I20260 on pSB3K3 inocluated into Kanamycin LB
strain list is updated with 2010 parts added yesterday
J23101 on J61002 from frozen stock is inoculated into Ampicilin LB
Transformed I0500 from 2010/plate3/20B into DH5-alpha, plated on Kanamycin LB Agar
inoculated all recent parts on strain list (except E1010) to fill up plasmid -20 stocks
The primary constructs of AIP sender and reciever has been cut, extracted and set ligated (O/N, 16 °C) to J23107.

Monday, July 18

Inocluated pSB2K3-I0500 into Kan-LB

Tuesday, July 19

Performed preparative digestions of J23101 cut with EcoRI + PstI & PSB3K3 also with EcoRI + PstI
These parts were ran on gel and gel extracted:
J23101 : 0.0515g
PSB3K3 : 0.0259g
Results were not that great. There was not enough DNA and bands were too faint. Also, ladder ran a little funny, may have been overloaded. We will need to redigest. This time a diagnostic gel will be run to see what went wrong.

Wednesday, July 20

LPS: Cveta is looking at the pFB10 sequence
Dan is looking at Parts Sequences
DS: hunting for 153bp fragment on larger scale with new batch of Mph1103I enzyme
Quantification: constructing pSB3K3-J23101-RFP by E+P digest
Assembly: Stage 1 is assembled but not checked by diagnostic digest. Broth inoculated for miniprep, growing.

Thursday, July 21

DS ligation: three attempts were made with different tubes of competent cells each. Attempts:
-ve ctrl failed
-ve ctrl ok, many colonies still on the transformant plate. 4 larger colonies were pathced and broth-inoculated.
3rd attempt made today, spread on amp plate, growing
pSB3K3-J23101-RFP construct inoculated from streak plate into broth. two streakplates are in the fridge.
gel was ran. wells:
Ladder (Fermentas 1kb plus, as always)
Preparative digest of J23107+I746101 construct
Same
Undigested control for preprative digest above
Ladder
Diagnostic digest of J23107+I746101 construct
Undigested control for diagnostic above
pSB2K3-I0500 (part from the kit)
wells 5,6,7 and 8 pic is here, the other half of the gel was exposed to UV only to cut out the top band from well 2 and 3 to be gel-extracted.
pSB3K3-J23101-RFP was already inoculated into broth by Leah yesterday. Dan miniprepped that and the remaining culture of I0500. George made frozen stock #106 out of pSB3K3-J23101-RFP

This is the diagnostic gel we ran on the vector (J23107 + I746001). The bands are as follows:
ladder
digest with (EcoRI and XbaI)
Negative control (no restriction enzymes)
Digest again (left over from preparative, since I had to increase the volume and it didnt all fit into the well)
The preparative looked the same. respective band excised+extracted.

Friday, July 22

DS lig 3 failed (abundant growth on both plates
miniprepped DS lig 2 (4 of them) and another culture of 3K3 RFP J23101.

Tuesday, July 27

Ran the /NspI+PstI digestions on gel (2uL + 0.5uL PstI + 0.5uL NspI + 2uL FD buff + 15 uL water)
Ladder
DS clone 1
DS clone 2
DS clone 3
DS clone 4
pSB1A2 (I746104 part)
Prepped parts list has been updated. The sequencing confirmations are still to come from Dan Barlow.
BW27783 recieved from UBC, inocluated into liquid culture.

Wednesday, July 28

Made frozen stock of BW27783 #107
digestion of 3K3-J23101-RFP preparative: (7uL DNA + 1uL EcoRI + 1 uL SpeI + 2uL FD Green Buff. + 9 uL water); diag (3 uL DNA + 0.5 EcoRI + 0.5uL SpeI + 1uL FD Green Buff. + 5uL water) pic of diag
digestion of 2K3-I0500 preparative: (7uL DNA + 1uL EcoRI + 1 uL SpeI + 2uL FD Green Buff. + 9 uL water); diag (3 uL DNA + 0.5 EcoRI + 0.5uL SpeI + 1uL FD Green Buff. + 5uL water) pic of diag the lower, 1200bp band is the target

Thursday, July 29

pFB10 inoculated into broth, Km+Sm
pFB10 streaked on Sm and Sm+Suc10%.
Transformed K359201-I0500 ligation, plated on Km agar.
FepA-Sensor construct was being miniprepped by Leah
Sensor-Generator construct inoculated into LB.

Friday, July 30

Planned for the upcoming week
Worked on the construction tree
Prepared for Outreach lab event

Tuesday, August 3

Sucrose sensitivity of pFB10 confirmed.
inoculated broth with 6 different colonies of K359201-I0500.
Finding source of microplates. According to this, the clear plates are even better than clear-bottom-black ones.
K201-I500 construct miniprepped, digested E+P, ran on gel:
Sample 1-1
Sample 1-2
Sample 2-1
Sample 2-2
Sample 3-1
Sample 3-2
Ladder
linky
matches expected results (from ApE) ideally.

Wednesday, August 4

21 plates for ESQ activity poured
~10mL of 10% L-arabinose stock created from Charles lab supplies (used 1g). Used protocol from OpenWetWare for creating stock.

Thursday, August 5

Made 0.2% arabinose LB agar plates.
Streaked K359201-I0500 construct on an Ara-LB plate.

Friday, August 6

DH5-allpha with K359201-I0500 shows no red phenotype.
Transformed ligation of P2-RFP (I746104+I13507)

Monday, August 9 - Friday, August 13

All attempts to transform that P2-RFP has failed
Autoclaved
4 baffled fasks with 50mL LB (freshly prepared, measured out with graduated cylinder)
two jars of µfuge tubes
100mL 0.1M MgCl2
2 GSA bottles
LPS: Inoculated 5 different cultures of pFB9010 into Km/Sm LB liquid media, as well as into blank LB media. These are needed in order to continue with the Landing Pad Project from 2009. The cultures will be used to make frozen stocks of the plasmid, as well as minipreps for future work. Results will be seen tomorrow. Previous attempts to grow the pFB9010 plasmid containing cells have been unsuccessful – although the inoculation is from Km/Sm patches, no growth has previous been observed in Km/Sm liquid cultures.
Amp plates were tested by using two plates – one Amp and the other Blank. On them were plated MT616 and DS plasmid. DS plasmid is Amp resistant, MT616 is not. Results to be seen tomorrow.

Monday, August 16

Missed the exponential phase for the comp cell prep, deferred till tomorrow.
LPS: There was no growth in the Km/Sm liquid cultures but there was growth in the blank cultures.
LPS: Two plates of pFB9010 have been recovered. In order to solve ambiguity, liquid cultures of Km and Sm were inoculated from plate 1 (most used). Also, plates of Km, Sm, Km/Sm were streaked with each of the 5 patches from each of plates 1 and 2 (where plate 2 is Sm only for some reason…although it should be both). This should solve which of the resistances is not functional. Results tomorrow.

Tuesday, August 17

Assembly: Nonodropping of both J23107 and I13507 stocks was performed. Digestion of J23107 with PstI and SpeI was unsuccessful. The 0.8% Agarose gel showed that the plasmid was not cut, since the uncut control beside it yielded the same bands.
Assembly: Digestion of I13507 with PstI and XbaI was successful, although the bands were slightly off as related to the ladder. Suggestion was made that Gene Ladder Plus should be ran as 2uL of solution + 8ul of water. Also, it has ran fast before, so beware. Bands expected are 883 and 2057 (from I13507 in pSB1A2 plasmid). 883 was taken (appeared at around 1000, however), since it is an insert. The bands were gel purified and stored at -20 C.

August 18

digested and gel extracted I13453/E+X and I13458/E+S
made 30 plates for ESQ activity
performed half of competent cell procedure, let incubate on ice in 4°C O/N
Results from yesterday were as follow:
Growth from both pFB9010 plates on Km and Sm, but not on Km/Sm.
Liquid cultures showed growth in Km, but not in Sm.
Not sure what this means at the moment, so further tests need to be done.
Transform remainders of pFB9010 stored at -20 C (nanodrop, transform)
Try digestion of J23107 again – did not work AGAIN! Will attempt another time but with another lab’s REs, SAP and buffer.
If digestion works, ligate with I13507. – did not work =(

August 19

Digestion of J23107 was re-done using REs, Buffer and SAP from another lab (except for SpeI, since it was not available from anyone else.) This did not work either.
We think that maybe there is something wrong with the J23107 miniprepped stock in the -20C fridge, so we will make a new one. Took J23107 from frozen stock and streaked onto an Amp100 plate, as well as inoculated into Amp100 liquid culture. This will be miniprepped tomorrow.
Set ligation of I13458+I13453

August 20

Miniprepped J23107
Nanodropped and digested – SUCCESSFUL!!!!
Gel Purified and Nanodropped
Concentration according to nanodropping is ~ 1ng/ul = way too low.
More J23107 and I13507 will be inoculated over the weekend, and will be minirepped and digested on Monday.
Regarding LPS, took a Km20 plate which yielded good results, and inoculated it into the following liquid cultures.
Km20, Km10, Km20/Sm100, Km20/Sm50,Km10/Sm100,Km10/Sm50, Sm50, Sm100.
This was done to ensure that the concentration of Sm or Km is not too high in the liquid cultures (mass transfer accounts for erroneously high concentration in comparison to solid agar on plates.)

August 22

Results from inoculations of various Km/Sm concentrations were as follows:
Contents Results
Km10 +++ (red precipitate on the bottom)
Km20 +++ (red precipitate on the bottom)
Sm50 +++(very little white-ish red precipitate on the bottom)
Sm100 -/+ (looks little bit cloudier than regular LB, but that could be due to re-suspension of the patches, since it was taken from a patch, not from a single colony)
Km10/Sm50 +++ (red precipitate on the bottom)
Km10/Sm100 +++ (no red precipitate on the bottom)
Km20/Sm50 -/+ (looks little bit cloudier than regular LB, but that could be due to re-suspension of the patches, since it was taken from a patch, not from a single colony)
Km20/Sm100 -/+ (looks little bit cloudier than regular LB, but that could be due to re-suspension of the patches, since it was taken from a patch, not from a single colony)
This probably means that while the resistances are present in the genome, they are not expressed very strongly. Will attempt to grow colonies on plates with reduced Sm/Km. After, will attempt triparental mating again.
innoculated J23107 into 5 liquid Amp 100 cultures, ready to be miniprepped tomorrow (Monday) prepared Km10/Sm50, Km10/Sm100, Km20/Sm50, Km20/Sm100 plates and streaked pFB9010 on them. Results to be seen tomorrow. This was done, since liquid versus solid media has been giving varying results with this project.

August 23

There is no growth on any of the pFB9010 plates (with varying conentrations of Km and Sm)
Miniprepped and nanodropped J23107 (concentrations are written on the tubes)
Digested J23107 with PstI and SpeI
Ran diagnostic + preparative gel
Gel extracted
Digested I13507 with PstI and XbaI
Ran diagnostic + preparative gel

August 25

Digestion of I13507 with PstI and XbaI could not be performed yesterday, so will be performed today
Ran diagnostic + preparative gel
gel extracted

August 26

Concentrated samples of digested J23107 (digestion was done with SAP) and I13507.
Performed ligation (added ATP)

August 27

Ligation was ran on gel (big mistake....should have just went along with transformation right away, since results could not be seen on the gel)

August 30

Performed digest of J23107 again, except used FD Buffer instead of Green FD Buffer
Ran on gel
Sample appears as a smudge (potential contamination)
Performed digestion of I13507 again, using FD buffer instead of FD buffer green.
Ran on gel
Could not see digest.
Transformed ligation of “58+53” (quantification work)

Tuesday, August 31 - Friday, September 3

Performed digest of J23107 and I13507 AGAIN! This time, SAP was not added to the vector digest (usually, if it is a double digest, you don’t really need to add SAP. It helps in the case that one of the REs does not cut). WORKED!!!
Transformed the ligation of J23107 and I13507
Using 50µL of competent cells, 20µL of ligation reaction and 15µL of CaCl2(100mM solution)

September 6 - September 10

Orientation week

September 13 - September, 17

First Lab meeting for Fall 2010
created a new iGEM active lab & a new thread of e-mail to keep everyone updated on lab work done daily
Innoculated I746104 and I13507 into liquid broth (3 different ones)

Monday, September 20 - Wednesday, September 22

Planned for Jamboree
Assessed previous work done and decided and what will be done for the competition
Organized a new schedule to get the work done accordingly for the jamboree
Miniprepped I746104 and I13507

September 23

1. Nanodropped samples of I746104 and I13507. Results are as follow:

I13507 - [102 ng/ul] 260/280 = 1.87
I746104 (1) - [168.8 ng/ul] 260/280 = 1.81
I746104 (2) - [158.8 ng/ul] 260/280 = 1.82

Calculations for digestions: need 617.9 ng of insert, 300ng of vector.

1. Digests:

I746104 with SpeI and PstI 3ul I746104 1ul Pst 1ul Spe 2ul FD Green Buffer 12ul Nucl. Free Water 1ul SAP
This was done in duplicate. Control: 1.5 ul I746104 without REs; 10uL reaction.
I13507 with XbaI and PstI 7ul I13057 1uL PstI 1uL XbaI 2uL FD Buffer Green 9uL Nucl. free water
control: 3ul of DNA, no REs, 10ul reaction.
3. Left to incubate for an hour @ 37C
4. Gel purified using protocol for purification from enzymatic reactions (used 60uL of Elution buffer for non-controls, 30ul for controls)
5. Speed vac for 10 minutes on high.
6. Nanodropped
I746104 (1) - 9.7 ng/uL
I746104 (2) - 8.7 ng/uL
I746104 (3) - 5.5 ng/uL
I13507 (1) - 14.1 ng/uL
I13507 (2) - 21.3 ng/uL
I13507 (contaol) - 14.0 ng/uL

7. Ligation

Vector 8uL
Insert 6.5 uL
10X lig buff 2.5uL
ligase 1.5 uL
ATP 1uL
Nucl. free water 6
total: 25.5 uL

The two I13507 were mixed, and 6.5 uL were taken from each into the 3 aliquots of I746104. Why is there 3 aliquots, when there was only 2 to begin with, you ask? Because Cveta lose the label on one of them, and couldn't tell which were the actual digests and which was the control.
To fix this grave mistake:Cveta saved 2uL from each of the three tubes so we can run a gel tomorrow and find out which is which. If you are doing anything with the tubes, please ensure the labelling does not change. Left to ligate at 16C in PCR machine.

September 24

Ligation of K359003 and I13507 was completed, plated on Amp100 and incubated overnight at 37˚C

September 25

Informed the lab volunteers of work done over the summer
Shared the following with them:
The Handbook

In Brochure form (please print with Acrobat Reader in the "short" duplex format, or otherwise I am not responsible for wasted paper)

September 26

checked the I746104 digestions that went into ligation by gel, #1 is apparently a control.
Transformed and plated the ligation (putative 003).

September 27

Incubated Amp100 K359003 plates grew well. Some bright pink colonies were visible, others were white.
Two aliquots of ligation #2 and #3 were prepared
3 pink colonies from each plate were inoculated from each plate into separate liquid broths of Amp50.

September 28

Miniprepped the 6 separate tubes, nanodropped, written concentration on side of the tube and stuck in second green box (the one labeled "2010 Parts Kit 2", disregard the meaning of that name), so it is easier to find.

September 29

6 clones were digested using the following gel layout:
1: Ladder (2uL + 8uL MQ)
2: clone #1 /E+P
3: clone #2 /E+P
4: clone #3 /E+P
5: clone #4 /E+P
6: clone #5 /E+P
7: clone #6 /E+P
8: I13507 /E+P

October 1

Attempted to make K359009, K359008, K359008 (3A), and K359009 (3A)
3A was not sucessful
K359008 : obtained from K359006 and K359003
K359009 : obtained from K359007 and K359003
Samples were digested, ligated and incubated for over 2 hours at 37˚C
All 4 samples were purified directly from the enzymatic reaction
Samples were nanodropped
SAMPLE ---> CONCENTRATION---> 260/280
K359003 --> 13.5ng/µL ---> 1.57
K359006 --> 13.8ng/µL ---> 1.90
K359003 --> 7.4ng/µL ---> 1.21
K359007 --> 45.7ng/µL ---> 1.78

October 2

1. Digests
Sad news of the day: SpeI is not heat inactivatable (sp?). Some NEB heat inactivatable SpeI was found, so we shall try that one later.
a. Digests # 1 to make K359008 003 cut with XbaI and EcoRI 006 cut with SpeI and EcoRI
b. Digest # 2 to make K359009 003 cut with XbaI and EcoRI 007 cut with SpeI and EcoRI

2. Ligations The above digests were set for ligation. Two aliquots were made for each (but there was barely anything for the second one.) A -ve and +ve control were also included, where -ve = no DNA, +ve = Amp resistant plasmid lying around. Left tubes in PCR machine overnight at 16C.

October 3

Transformed and plated the tubes from yesterday on Amp. They were left in the 37C incubator overnight.
Reinnoculated DB 3.1

October 4

Researched what to do if we ran out of linearized plasmid pSB1c3
http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones
http://partsregistry.org/Help:Spring_2010_DNA_distribution
Decided to leave 3A alone for now
http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly

October 5

Checked on the plates from Saturday:

-ve ctrl : no growth
+ve ctrl: no growth
009-1: contaminated
009-2: some rosy colonies and white ones. inoculated 5 rosy ones into tubes, but they were so close to each other, I also made one streak plate as backup
008-1: few rosy (and white) colonies
008-2: (white), rosy and bright red. went for bright red, 5 tubes, 2 streak plates
DB3.1 reinoculated (again)
LEFT:

11 tubes on shaker
3 plates in fridge
3 plates in incubator

October 6

Transformed the DB3.1 :
http://partsregistry.org/Help:Spring_2010_DNA_distribution pSB1C3-BBa_P1010 from Spring 2009 Distribution Plate 1 Well 5E

2009 plate 1 was obtained,
10uL MQ was pipetted into Well 5E and all was transfered to a microfuge tube. *labelled: 2009-1-5E, pSB1C3-P1010 1mL of DB3.1 culture was pipetted into microfuge tube. Spun down (~12,000rpm/2 min), decanted and kept on ice. This was repeated twice with 1mL of CaCl2 but spun down in the fridge with the glass door.
50uL of CaCl2 + 2uL of DNA from the 5E well were added and tube was kept on ice for 30 mintues.
Transformation was proceeded as would normally be done using the protocol.

October 7

Transformation Results: FAILED. No growth on positive control or negative control.
Reinnoculated DB3.1 culture. Transformation will be reattempted.

October 8

Attempted transformation again using same protocol as before.
DIGESTION OF 008 & 009 PERFORMED AS INDICATED BELOW:
008-4,5(older prep),7(newer prep) will be digested E+P and ran on gel :
Ladder
008-4 /E+P
008-5 /E+P
008-7 /E+P
006 /E+P
008-4 undigested
008-5 undigested
008-7 undigested

Monday, October 11

Transformation FAILED. This will not be redone. To be discussed in weekly meeting on thursday.
009-5 was speedvaced,digested with E+P and ran on gel
Worked on updating the Wiki and presentation

Tuesday, October 12

Analyzed results from gel on friday:
Ladder: yeah.
008-4 /E+P: ladder contamination?
008-5 /E+P:
008-7 /E+P: the most probable, don't ask me what is in the lowest band
006 /E+P: overkill amount, had to have lower exposure to view
008-5 undigested: looks fine for undigested
009-5 E+P: see above
007 /E+P: overkill

Wednesday, October 13

Worked to complete Human practices section
Individual photos of active team members were gathered with a small blurb about each person for wiki
Worked on updating Quantification for the lab wiki & the SVG tree

Thursday, October 14

Board meeting: updated on work done in all sections, upcoming work plans made
Weekly Lab meeting: shared information from board meeting with the rest of the members and accordindly, created an agenda:
Wiki: all due Saturday morning by 12 pm (Oct 23)
Lab:
Some intro (very general)
Quantification: (by Monday, Oct 18)
Construction tree- SVG (by Monday, Oct 18)

Monday, October 18

Team photos taken
Looked through our fridge and freezer. Put all relevant microfuge tubes into the "2010 Parts Kit 2" green box.
Could not find 006, nor 007 but did find 008 and 009 and so,inoculated 3 tubes for each.
Digested parts 003, 006, 007, 008, 009 and pSB1C3 with E + P. Purified these digests and then left them over night in part + pSB1C3 ligations. Also, made 3 inoculations of pSB1C3 and 2 inoculations each of 009/008 (total of 7). These are all on the shaker.

Tuesday, October 19

Miniprepped pSB1C3/008/009 (9 tubes in total) from the incubator.This is required in the case that the transformations are unsuccessful,so that we can start over.
Nanodropped them and results are as follows:
pSB1C3 - 1
CONCENTRATION: 48.7ng/uL
260/280 1.96
pSB1C3 - 2
CONCENTRATION: 185ng/uL
260/280 1.91
pSB1C3 - 3
CONCENTRATION: 58.4ng/uL
260/280 1.93
008-1
CONCENTRATION: 59.8ng/uL
260/280 1.75
008-2
CONCENTRATION: 131.7ng/uL
260/280 1.86
009-1
CONCENTRATION: 121.7ng/uL
260/280 1.95
009-2
CONCENTRATION: 209.0ng/uL
260/280 1.92
Transformed all 5 ligations, which were in the PCR machine, in DH5alpha on to Cm plates. Stored the remainder of the ligations in Parts Kit #2, green box in freezer.
Made media broth - 200mL, placed into 5ml tubes and autoclaved.

Wednesday, October 20

Ligated 006 into pSB1C3..? RESULTS: TO BE UPDATED
Cut out the RFP which is currently with the pSB1C3 (5 tubes labelled pSB1C3 contain that plasmid plus an RFP biobrick.
We need to cut out the RFP biobrick and leave just the plasmid for ourselves.
Need to do the insertions of the biobricks into the desired plasmid (pSB1C3).

Thursday, October 21

Worked to complete Human Practices section
Continued work on Jamboree Presentation. Work will be continued on the weekend.
streaked and inoculated into liquid media.

Friday, October 22

Miniprepped the inoculations from yesterday and nanodropped them. Results are as follows:
These need to digested and shipped off to Boston.
Still need have lots of work pendning for the presentation and other administrative work that will be worked on the weekend and carried over for next week.

SAFETY

Laboratory Safety

The Ribozyme Project is not expected to raise any research, public or environmental safety concerns other than those normally associated with Biosafety Level 2 organisms, such as Escherichia coli (DH5-alpha), which is classified as very low to moderate. The use of this project is primarily reserved for research and laboratory use, therefore, should not purposefully be exposed to the public or environment except after further testing in its specific applications (such as with particular fusion proteins). Furthermore, the basis of our project is to establish a self-excising sequence (ribozymes), which should limit the expression of any intervening sequences to the RNA level. If the intervening sequence were something of environmental or public relevance (such as antibiotic resistance), the experimental design indicates that the sequence will be removed and, thus, not expressed. This is a relevant contribution of the design in limiting expression to the RNA level, which eases environmental hazard concern upon the accidental release of a GMO containing this biobrick. Therefore, the new biobrick parts submitted should not raise any safety issues.

The necessary facility, equipment and handling procedures associated with Level 2 Biosafety concerns were met:

1.Pipetting aids
2.Biosafety cabinets where applicable
3.Laboratory separated from other activities
4.Biohazard sign
5.Proper safety and disposal equipment, including autoclave
6.Personal protective equipment, worn only in the laboratory
7.Screw-capped tubes and bottles
8.Plastic disposable pasteur pipettes, when necessary

All precautions with respect to recombinant DNA were observed:
1.All waste was autoclaved before being thrown away.
2.Researchers practiced aseptic technique and personal hygiene and safety precautions
3.Procedures likely to generate aerosols are performed in a biosafety cabinet
4.Bench surfaces were disinfected with ethanol.
4.Potentially contaminated waste is separated from general waste

Safety Questions

1. Would the materials used in your project and/or your final product pose: The materials used in the lab are non toxic to health of individuals as well as to the environment. One of the major reagents that is used is GelRed which is used as a substitute for Ethidium Bromide. Gel Red is unable to penetrate into cells and so is a non-mutagenic agent. As well it has the same spectral characteristics as Ethidium Bromide and so has the same effectiveness of use. The project itself is safe even if released into the environment by design or accident since the part being expressed is the Green Fluorescent Protein (GFP). Unless the sequences are mutated, the project poses no risk.
Please explain your responses (whether yes or no) to these questions.
Specifically, are any parts or devices in your project associated with (or known to cause):
- pathogenicity, infectivity, or toxicity? No
- threats to environmental quality? No
- security concerns? No
The parts that are associated with the project this year are at the same level of risk as the any of the regular parts that already exist. All parts are constructed in an antibiotic containing backbone so that accidental release of will pose minimal risk to contaminating other bacterial populations.

2.Under what biosafety provisions will / do you operate? a.Does your institution have its own biosafety rules and if so what are they? The University of Waterloo had a Bio-Safety plan in place to ensure the proper use to bio-hazardous materials in teaching and research at the university. A more detailed overview of their plans is outlined at the Bio-Safety Website
b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? The laboratories operating at the University of Waterloo have obtained permits from the Bio-Safety Committee in order to perform intended research. Since the Waterloo iGEM team performs all laboratory work in a parent lab under the guidance of the Masters and PhD students of that lab, the projects carried out in the lab are covered by the permits obtained by the parent lab.
c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training. All lab volunteers are required to take an online training to familiarize themselves with the Biosafety practices of the University of Waterloo. The training is followed up by a quiz ensuring proper understanding of the material. Upon completion of the training and quiz a hands- on lab training is provided under supervision of the parent lab’s PhD student. The hands-on training involves instruction of use of the appropriate equipment that is used in the lab, as well as how to maintain and discard materials in a safe manner.
d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible. Canada operates under the guidelines set up by the Public Health Agency of Canada. The Agency is the national authority on matters concerning biosafety and biosecurity. Risks to the public are reduced by standardizing controls over activities that involve human pathogenic agents, domestic or imported. While these guidelines are in place the current iGEM project does not involve work with any agents or materials that may pose a risk to humans. The link to the Public Health Agency of Canada is provided below: Public Health Agency of Canada