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.

University of Waterloo: iGEM OUTREACH

The purpose of UW iGEM Outreach has always been and will continue to be the connection between our community and us. To help build a better understanding of synthetic biology, how it has affected the world around us and to create a basic, fundamental knowledge of the subject that can be incorporated into the way we see things. That perspective can be positive or negative, but the point being is that we have provided that baseline knowledge that will allow our community members to form a fact-based opinion, meaning they are informed. This year, UW iGEM pursued two different outreach events, both on a grand scale. We have included some downloadable materials if you are interested in conducting your own workshop!

Grade 12 Outreach Workshop: March 25th, 2011

The first was an organized Grade 12 workshop aimed at biology students to gain a better understanding of synthetic biology, the industries it has been affecting, career prospects as well as two hands-on activities. Over the planning span of 3 months, this event was organized in close accordance with the Kitchener-Waterloo school board and with the Marketing and Recruitment Co-ordinator for Science at the University of Waterloo. Through the creation of a brochure and meeting with individuals from the school board we had sent out an invitation all across the district for students to come in for our event. Eventually we had gotten more than 85 students to attend, which was great as it was the first time we had implemented this idea.

The next step had then been to recruit interested volunteers for the event so we could have our own students give a helping hand and who shared the same passion of sharing knowledge as we did. In order to facilitate this we had sent out emails, gone to various lectures and talked to students all over campus to get them involved. After recruiting 12 volunteers the brainstorming process had begun. We had wanted to have an interactive workshop where students were not just listening to us talk, but were actually involved in a stimulating activity that they could be excited about. The first part of our presentation looked into what was synthetic biology, what were Genetically Modified Organisms (GMOs) and what were the positives and negatives of them. Once that was through we started the first event. The first event we had was to talk about our very own Canadian genetically enhanced Yorkshire pig called the EnviroPig™. It has the capability to digest plant phosphate more efficiently than traditional Yorkshire pigs, which do not contain the enzyme to break it down, phytase. This gene can be found in E.coli which had been inserted into a pig embryo to allow it to produce phytase in its salivary glands. This is a real technology in Canada and is currently a very hot topic of debate among many citizens, so we thought it would be ideal to introduce students to the world of biotechnology right in their very own backyards. Once students had been exposed to the information, we had given them a package which we had compiled giving the positives and negatives of societal, technological, ethical, environmental and economical issues.

Of course, it did not end there! Students were then put into groups of 5 and as mentioned above, one of the key goals of UW iGEM Outreach is to allow members of our community to make informed decisions based on facts. Supplied with markers and paper students were to give a 1-2 minute presentation on why they did/did not believe that the EnviroPig™ should continue receiving funding from the government of Canada. The best and most convincing presentation won- we had great discussions from the negative aspects to positive aspects to even a compiled rap song about the EnviroPig™!

After the first event we continued on with our presentation. Here we discussed current industries that had been affected by synthetic biology. This incorporated the pharmaceutical and biofuels industries specifically. We discussed how pharmaceuticals had incorporated an aspect known as biosynthesis where they could customize and fine-tune certain pathways using standardized parts in order to provide efficient and precise drug delivery systems. In biofuels we talked about first and second generation biofuels and how the use of synthetic biology has the ability to create a ‘superbug’ to look for corresponding metabolic pathways for yielding ideal results. After this we did the second activity.

The second event that had been implemented was to incorporate what we do in our labs, outside of the lab. Essentially, we wanted to introduce the idea of synthetic biology to students and how it was an extension of what we knew as genetic engineering and consisted of students not just from science but from math, engineering and computer science. The activity called, ‘Design Your Own Pathway’ gave a series of scenarios we had given students with a library of BioBricks to create a certain pathway. Progressively each scenario was harder, more complicated and required the use of multiple BioBricks. The BioBricks that we had used were from the library and were real parts. Essentially this was to enable students to have a feel of how we have a ‘mix and match’ concept when it comes to synthetic biology. The activity had been set up as a relay race, where students in the same teams as the previous activity had to race each other to finish all scenarios. The activity and concept had been such a success that after the workshop teachers had asked to use our activity in their own classrooms.

Finally, we completed the presentation with a discussion of career paths that students could pursue that did not necessarily involve working in a lab. We wanted students to understand that the world of synthetic biology and biotechnology encompasses the involvement of individuals from multiple backgrounds and can penetrate into many different industries.

There was such an interest in our workshop that our prospects for the next one are aimed at more than 150 students. One thing’s for sure, mission accomplished and there’s definitely more to come! Would you also like to have your own workshop at your high school or university? Please feel free to view the downloadable materials for the presentation and two activities or contact us at uwigem.outreach.hp@gmail.com.

Engineering Science Quest: July-August 2011

Founded in 1990, the Engineering Science Quest or ESQ is a not-for-profit program that operates with the goal of exposing children in the Kitchener-Waterloo region to the world of engineering, science and technology through engaging them in a variety of hands-on activities. Promotion is primarily done through workshops in-school but also have satellite programs which reach out to rural and Native communities as well.

This is not the first time that UW iGEM has been involved in ESQ and we are proud to say that our continued involvement has allowed us to develop a standard set of activities which we are pleased to present to kids ranging from Grades 3-6 with more than 100 students. Currently we are also developing ideas for older kids that are similar to our activities from the Outreach workshop we had in March for Grades 10-12 and for even younger kids from Grades 1-2. Through involvement with managers specifically for ESQ this year we were able to have continual workshops every week from July 11th- August 12th, 2011. This was done with the recruitment of volunteers who again shared the same passion as we did in connecting with our community to facilitate that baseline knowledge; to get students introduced or even extend their knowledge on the world of synthetic biology and biotechnology.

The first activity for Grades 3-4 was called, “All About Bacteria: Do You Really Need to Wash Your Hands?” In this activity we introduce kids to the idea of biology, bacteria, synthetic biology, and iGEM. We also introduce them to basic ideas of sanitary techniques and tools used in a standard lab such as petri plates, agar, swabs etc. and how to layout an experiment; what is your hypothesis, results and conclusions? Once we discuss these basic concepts we allow the kids to take a swab of their hand and plate it on half of a petri dish. They then clean their hands with sanitizer and swab the other half of the petri dish. They then receive another petri dish where they can swab other places to find other ‘neat’ bacteria that may be lying around on the floor, counters, door knobs or wherever else they want (except up their nose, in their eyes, ears or mouth!). At this point and throughout the activity, interaction with the kids is key, as they always have stories or thoughts and experiences that are enlightening to share- even to us university students.

Once we explain what incubation is, we allow kids to come in the next day and see all the different types of bacteria that may have grown on their petri plates and come up with conclusions from the results they have seen. We provide them with a record sheet where they are able to write down what they saw such as; morphology, colour, its surface and even elevation. To be able to interact with kids at such a young age and to introduce them to concepts which students are normally exposed to in higher grades, allows them to explore a part of their world that they never knew existed. Building curiosity at a basal level helps us connect with our community and bridge the gap towards making more informed decisions.

The second activity for Grades 5-6 was called, “DNA Extraction from Your Cheeks”. This activity centers around the idea of DNA, where it is found, what it looks like and how every living organism contains very similar genomes, proteins and enzymes. There were four steps to this process, first to collect cheek cells, second to burst cells open to release DNA, third to separate DNA from proteins and debris and finally isolate the concentrated DNA. Kids obtain a cup of Gatorade containing a saline solution and swish the drink in their mouth for about a minute while gently chewing on their cheek cells. Then detergent is added to the test tube and meat tenderizer is added and the tube is inverted gently a couple of times. Cold rubbing alcohol is then added with a pipette which should allow the DNA to be visible. Then kids transfer the DNA into a PCR tube where they can hang it on a string to make a really neat necklace.

Kids were keen and interested in these activities, especially learning about various different concepts that they had not yet been exposed to. “Who knew that DNA could come out of your own cheeks?” was the idea we wanted to pass along- that every living organism contains DNA. From bacteria to animals.

Are you interested in doing these activities with kids around this age? Feel free to contact us at uwigem.outreach.hp@gmail.com. It is an inexpensive, interactive and fun way to have kids involved in genetics, microbiology and synthetic biology at a very early age.

Introduction

Synthetic Biology has been perceived as a field of science that has introduced tools, concepts and technologies which are sometimes sociologically and ethically insupportable. The non-scientific community has developed a stigma against the application of various synthetic biology technologies. The problem does not lie within the technology, but by an institution’s poorly executed strategy in educating the prospective end user about the relative benefit of its product. At times, the rate of technological advancements in the bioengineering industry surpasses the end-user’s ability to understand the necessity of the technology.

The science behind these synthetic biology tools has been the current driver in instilling a sense of appeal and intrigue within the scientific community. However, it is critical that the institutions that develop these technologies educate the non-scientific community of the importance and benefit of these tools to help relieve these evolving stigmas.

This year, the University of Waterloo’s Human Practices team’s focus was to develop a marketing strategy for their 2010 design project, the Staphiscope. The Staphiscope is an enhancement to the existing conventional plating methods that are used to detect Methicillin Resistant Staphylococus Aureus.

The scope of the analysis includes:

What is the selling point of the product? What differentiates the Staphiscope from what already exists and is accepted by the non-scientific community?
Does the concept of competition exist within synthetic biology? If so, how does an institution market the Staphiscope to optimize their opportunities for growth?
How can the marketing strategy approach be differentiated to ensure that both the scientific and non-scientific community are exposed to the relative benefits of the Staphiscope?
How do we channels the knowledge and learnings acquired within the synthetic biology industry and channel it to other stakeholders that will prospectively be impacted by the technology?
The purpose of the analysis is to ensure that the scientific industry understands the importance of bridging the scientific theory of a product to its economic feasibility. Moreover, it encourages institutions who participate in developing synthetic biology technologies to align their perception of a revolutionary design to what will be socially acknowledged by a non-scientific community.

What is the Staphiscope?

Methicilin resistant Staphylococcus aureus (MRSA) are bacteria whose presence has been quite problematic in terms of human pathogenic infections. Since its discovery in the 1880s, Staphylococcus aureus has been the known cause for several kinds of minor skin, and major post-surgical infections. Prior to 1940, mortality rates in relation to this pathogenic organism reached 80% (Deurenberg and Stobberingh, 2008.) The first wave of resistant S. aureus came two years after the introduction of penicillin for medicinal practice in 1940. In less than twenty years, most of the S. aureus strains known to man were unaffected by the antibiotic. In the late 1950s, a penicillinase-resistant penicillin was brought forth to the medicinal market; this was methicilin (Deurenberg and Stobberingh, 2008.) The bacterium's subsequent resistance to methicilin came two years after its introduction in 1959. The mecA gene responsible for methicillin resistance is also to blame for the organisms’ desensitization to several classes of antibiotics (Deurenberg and Stobberingh, 2008). This has led to the creation of the term: MRSA, now commonly used worldwide.

While an MRSA infection is much like a S. aureus infection, the difference comes in the lack of sensitivity of the MRSA to several classes of antibiotics. This makes MRSA infections a serious threat in both health and economical aspects.

The infection could be easily transferred directly (through regular skin-to-skin contact), as well as indirectly (through contamination of surfaces). The ease with which infection could occur, as well as the high mortality rate involved with the infection, make MRSA a serious threat to the health of patients worldwide (Durai et al., 2010.) There are only a few methods, which currently exit for MRSA screening. These are plating, liquid-broth inoculation, and PCR (polymerase chain reaction) assays. While these will be discussed in further detail later on in the report, it is important to note that these methods could be costly and time consuming, and could sometimes present incorrect results (Durai et al., 2010.)

In 2010, the International Genetically Engineered team at the university of Waterloo had been working on a method which can be used in supplement to the already-existing ones for the detection of MRSA infections. It is a proof of concept for diagnosing the presence of Staphylococcus aureus in an infection site before it has had the chance to create an infection. This synthetic biology–based diagnostic method will take advantage of the quorum sensing mechanism of Staphylococcus aureus, and utilize Escherichia coli as the sensor and reporter. For more information with regards to the Design of the Staphiscope please see the University of Waterloo’s 2010 iGEM Page: https://2010.igem.org/Team:Waterloo Who are the “Competitors”?

Treatment of an S. aureus infection is of extreme importance, as outcomes can range from simple pustules to death. Early detection and treatment of S. aureus carriers can reduce healthcare costs and greatly improve the health of the patient. There are only a few methods currently available to hospitals for the diagnosis of S. aureus infection. These methods are time consuming, expensive and, in some cases, not very reliable. The purpose of the Staphiscope is to provide the option of accelerating the method of detecting S. aureus, without compromising reliability. It will be a particularly useful tool in speeding up the conventional methods of serial plating.

The following section analyzes the current products on the market and compares them to the Staphiscope. BD GeneOhm StaphSR

Traditional culture methods for the detection of S. aureus are usually 2-3 days. The new BD GeneOhm™ StaphSR delivers a diagnosis in less than 2 hours, using rapid real-time PCR. Also, it can simultaneously detect and differentiate Methicillin-Resistant S. aureus and S. aureus from a positive result (blood culture). Currently under development are additional specimen claims – nasal and wound samples. Nasal claim will be able to determine colonization status and the wound claim will add to the rapid identification of positively infected patients. Obtaining positive results in a timely fashion aids clinicians in the prevention and control of infections.

Method

Specimen is prepared by transferring an aliquot of positive blood culture into a buffer team and then vortexed at high speed.

Cell suspension is lysed (via vortex and centrifuge) and heated. Then the lysis is cooled. Molecular reagents are reconstituted and the sample undergoes PCR. Results are obtained in about 1 hour.

References

"BD - GeneOhm - Products." BD: Medical Supplies, Devices and Technology; Laboratory Products; Antibodies. BD, 2011. Web. 23 July 2011. .

GenoType MRSA

Genotype MRSA uses DNA Strip Technology to identify both S. aureus and S. epidermidis cultures. Therefore, coagulase-positive and coagulase-negative staphylococci are differentiated. Simultaenously, the product can also detect Methicillin-Resistant S. aureus and S. aureus , which is incredibly important for therapeutic purposes. The test is performed from an overnight culture and guarantees results in only 4 hours.

Method

Culture is isolated from a sample or a primary culture is used.
DNA is extracted via methods producing amplifiable DNA from bacteria (for example QIAamp DNA Mini Kit from Qiagen).
Nucleic acids are selectively replicated in an amplification reaction. In the next step, the amplicons are chemically denatured, since detection on the DNA•STRIP® is done using single-stranded DNA. During the conjugate reaction, the specifically bound amplicon is marked with the enzyme alkaline phosphatase and is then made visible in a colorimetric detection reaction. In this way, a specific banding pattern develops on the DNA•STRIP®. Using a test-specific evaluation template, the test result can be read out quickly and clearly.

References

"GenoType® MRSA | Identification of MRSA from Cultured Material." Hain Lifescience | Ihr Partner in Der Modernen Labordiagnostik. Hain Lifescience, 2011. Web. 23 July 2011. .

"DNA•STRIP® Technology." Hain Lifescience | Ihr Partner in Der Modernen Labordiagnostik. Hain Lifescience, 2011. Web. 23 July 2011. .

Brilliance Agar

Brilliance Agar is a chromogenic screening plate used for the diagnosis of Methicillin-Resistant S. aureus. It uses a novel chromogen to show a blue colour when Methicillin-Resistant S. aureus phosphatase activity. For sensitivity, it contains antibacterial compounds which inhibit growth of various competitor bacteria. This product has high sensitivity and specificity, which minimizes healthcare costs. However, results are presumptive and would need to be confirmed. Organisms with atypical resistance can give false negative results.

Method

Patient is inoculated using a screening swab or an isolated colony or liquid suspension is used. Sample is plated on Brilliance Agar. Plates are incubated at 37 ºC. Results are obtained in 18 hours. Methicillin-Resistant S. aureus grows as denim blue colonies.

References

"Brilliance MRSA AGAR." Oxoid - Worldclass Manufacturer of Dehydrated Culture Media and Diagnostics Products. Oxoid, 2010. Web. 23 July 2011. .

Competitive Analysis Matrix

Factor StaphiScope BD GeneOhm StaphSR Brilliance MRSA Agar GenoType MRSA Resource Use 2 1 1 1 Extra Features 1 2 1 2 Reliability 3 3 1 1 Ease of Use 3 2 1 2 Time 2 3 1 2 Sensitivity 3 3 3 1 Appearance/Image 3 2 2 3 Sales Method 3 1 1 2 Cost 3 1 1 2 Total 23 18 12 16

Rating Scale FACTOR 1 2 3 Resource Use High Medium Low Extra Features Few Medium Many Reliability Low Medium High Simplicity Low Medium High Results Time High Medium Low Sensitivity Low Medium High Appearance/Image Poor Medium Excellent Cost to Buy/Use High Medium Low

Methodology for Rating Scale

The Competitive Analysis Matrix uses a format that divides the product’s market profile into several categories, providing a framework that ensures all issues are considered. Each category is scored in the course of the ratings process (using a numerical scoring system). Products are scored 1 through 27; those facing greater competitive threats would wind up with an overall low market profile score. Please note that ratings represent, in the end, an opinion. Please note, thorough assessment of each product’s market profile requires a broader framework, involving a comprehensive review of the product’s competitive position. Conclusions

Based on a comparison and competitive analysis of the Staphiscope and the three major detection products on the market, the Staphiscope was found to have the highest market profile score. It uniquely scored highest in reliability and ease of use. Although all products on the market signify great innovation in the field, Staphiscope has great potential to gain market share.

Porter’s Five Forces analysis

Marketing Strategy

Pitching the Staphiscope to a Research and Development Company When pitching the Staphiscope to the R&D industry, it is of paramount importance to establish its competitive advantage over traditional methods for S. aureus detection. To have a competitive advantage, a product or company must excel in at least one of the three following categories while remaining equal to its competitors in the others (Szarka, 2010):

Market access
Infrastructure
Technology

Market access refers to how easily the target demographic can get the product. Even a revolutionary technology can be hampered by a lack of market access. Infrastructure is related to whether or not a supply chain exists, as well as facilities for production and distribution. The third point, technology, is where the Staphiscope excels; its superior technology is what will separate it from competitors. Other selling points of the Staphiscope include its speed, efficiency, and room for expansion (could this technique be applied to other cases?). Conversely, there are also barriers that must be overcome when pitching to the R&D industry; namely, one must avoid falling prey to “not invented here” syndrome (Szarka, 2010). This term applies to the mindset some companies have of only trusting in-house innovation, and can be difficult to alter. Other barriers include the lacking of Staphiscope use on a commercial scale, and public perception regarding E. coli. While the latter issue can be combated via a public awareness campaign, the former is simply a fact of any innovative technology.
Companies such as Amyris Biotechnologies and Codon Devices have attempted to commercialize in the field of synthetic biology. Their experiences provide valuable lessons that can applied to the Staphiscope (Wilan, 2005). The most relevant of these is that patent protection should be a serious concern when developing a novel technology, especially in such a burgeoning and competitive field. Consolidating IP ownership and control early is equally important, as doing so will prevent a lack of unified vision from stalling the Staphiscope’s progress. Of course, there are a plethora of patent and IP issues surrounding the field of synthetic biology that must be dealt with before or during the commercialization process. Chief amongst these is the scope of protection that can be provided to new technologies (Chugh Sakushyma, 2009). Additionally, looking for a pharmaceutical partner can help ease the transition into commercialization.
It is important to realize that commercialization is a multi-year effort. As such, it can be beneficial to develop a commercialization plan (Office of Technology Transfer). This plan gives a general outline of what information the R&D industry will be looking for when it comes time to make a pitch. Details about market size and growth rates, predicted sales figures for the first five years, licensing agreements and more can all prove invaluable when attempting to bring a product to market. Preparedness in regards to this information can make the difference between a successful pitch and an unsuccessful one.
In summary, pitching the Staphiscope to the R&D industry is a complex process that must be handled correctly to ensure its successful commercialization.

References

Office of Technology Transfer. Argonne National Laboratory. “Commercialization Plan Worksheet”. http://www.anl.gov/techtransfer/pdf/LicensingQuestionaire1-0.pdf

Wilan, K.H. Nature Publishing Group. 2005. “Commercializing synthetic biology”. http://www.nature.com/bioent/startup/072005/full/bioent870.html

Chugh, A., Saukshyma, T. 2009. “Commercializing synthetic biology: Socio-ethical concerns and challenges under intellectual property regime”. http://stopogm.net/webfm_send/315

Szarka, M. “Adventures in Commercialization”. 2010. http://webcast.utm.utoronto.ca/1/watch/515.aspx



How Do We Achieve Public Acceptance?

For years, it has been believed that the prevalence of MRSA infections has resided within the walls of either a hospital or health care facility. Due to understated hygienic practices, a modified and relatively more virulent strain of Staphylococcus Aureus has also threatened several community based settings. In addition to Hospital Acquired MRSA, general health practitioners must also take into account the occurrence of community acquired MRSA. It is important to educate the public about the applications and relative benefit of the Staphiscope. Thus, there needs to be a channel of communication that allows for a transfer of knowledge between the scientific and non-scientific community.

Who Do We Want To Educate?
Given that MRSA may not only be spread within a hospital but also within the community, there are quite a few demographics that would gain value in understanding the benefit of the Staphiscope.
Hospital Setting
At least 2% of patients in hospitals or health-care facilities are likely to carry a strain of Staphylococcus Aureus that is resistant to antibiotics such as Methicillin. The following individuals are susceptible in contracting an MRSA infection:

Individuals who have weak immune systems
People receiving kidney dialysis or cancer treatments
Individuals who have been hospitalized or have had surgery over the past year
MRSA within the Community

Patients who are discharged may have an inactive form of MRSA that remains colonized in their system. Thus, those who live within the community are still at risk of acquiring and suffering from antibiotic resistant Staph infections that are relatively more virulent. MRSA has been detected and acquired by healthy people in both institutionalized and individual based settings. The following individuals are susceptible in contracting MRSA in the community:

Day-care facilities or facilities in which equipment of personal items are shared
Military bases
Individuals who get tattoos

The Strategy

In the field of synthetic biology, there is an unrecognized paradox that hinders the level of interdisciplinary communication between the scientific and non-scientific community. The stigmas built outside the realms of a scientific community grow at a greater rate than a scientist’s ability to answer the plethora of questions surrounding the regulation, safety and potentiality of these synthetic biology technologies. Another problem lies in a scientific institution’s inclination to address the ethical concerns surrounding their synthetic biology technology or process. Generally an individual who has not be previously exposed to the field, will construct an ethical judgement proceeding the phase where a company is about to commercialize their product. At this critical stage of a company’s product plan, stakeholders are reluctant in addressing these ethical concerns as it may discourage their ability to advance and innovate their existing technology. Regardless of the threats that these ethical judgments have previously imposed, there is a necessity in developing an approach in which the end result is an internationally renowned sense of public acceptance and support. “Without public support and understanding of research into synthetic biology, both funding and regulation are unlikely to support significant scientific advances” (Gaisser, et. al, 2009). To address this issue, an expert committee in Europe supported by the New and Emerging Science and Technology programme set out a roadmap that outlines the four interconnected attributes that will instil a sense of public acceptance within the non-scientific community. These four attributes are:

Scientific Milestones
Knowledge Transfer
Funding
Regulation

This roadmap outlines that without the implementation of a knowledge transfer strategy, there is limited confidence attaining in achievements under the other three key areas of the model. Scientific milestones would not be achievable without a sustained level of funding provided through other groups impacted by the prospective commercialization of these synthetic biology tools. Moreover, regulation of synthetic biology tools is influenced by the ethically themed perceptions created by both the scientific and non-scientific community.
The knowledge transfer strategy would be an interdisciplinary model that would impact the following stakeholders

Natural and Social Scientists
Engineers
Industry Representatives
Non-governmental organizations
Non-scientific community (ie. hospital staff, hospital patients and members within institutionalized settings)
Decision makers (ie. potential investors)
Funding Agencies

Knowledge Transfer Strategy

In this context, knowledge transfer is the exchange of research knowledge between the institution producing the synthetic biology technology and their target end-user. The purpose of the knowledge transfer strategy is to develop a communication platform between all stakeholders to mitigate the unsourced perceptions, bias’ and stigmas against the field of synthetic biology. Moreover, it is better to develop an interdisciplinary team at the early stages of the technology development phases versus segregating the two communities until the technology is prospectively in its commercialization phase.

Method

The following are examples of methods that can be used by the synthetic biology industry in promoting tools such as the Staphiscope to its potential end-users (ie. individuals within hospitals and community facilities) Development of an interdisciplinary network that consists of synthetic biologists, engineers, potential investors, and the general public. Sustainable dialogue between all stakeholders within the interdisciplinary network. This can be achieved through: Workshops that aim in exchanging information to individuals that will benefit from the application of the Staphiscope in either a hospital or community based setting Educational materials that are specifically tailored to the different parties that could be impacted by the commercialization of the StaphiScope (ie. Hospital Microbiologists, Patients, and the General Public) Incorporating the General Public and other stakeholders at the research phase of the technology development process. This results in a mutual benefit to both the institution as well as the end-user in that they are able to collaboratively discuss the ethical feasibility of the technology.

Use of knowledge broker- A knowledge broker is essentially an intermediary resource that assists in translating research knowledge to information that is comprehensible to a member that is not from the field of synthetic biology. An individual that has attained perceptions, knowledge and experiences from the scientific, business and social field is best suited to convey the importance of the Staphiscope to the stakeholders that have been listed above.

Partnerships between Synthetic Biology institutions and hospitals or other community based facilities.

In conclusion, synthetic biologists must consider the importance of implementing an approach to help relieve the prospective stigmas that could potentially impact the relative success of their technology. The general public plays an influential role in the innovation capabilities of these synthetic biology technologies. Thus it is critical that these synthetic biology institutions involve the public and create awareness of their technologies at all stages of the research and development stage. Integrating a strategy that incorporates the exchange of knowledge is a fundamental leverage that would help create an aura of public acceptance and support all dimensions of the synthetic biology roadmap.

Conclusion

The purpose of this analysis was to understand the relative feasibility of a synthetic biology tool from the perceptions of a scientific and non-scientific community. When devising a marketing strategy, most companies define a technology’s commercial success by its appeal towards investors or its ability to enter a market heavily saturated by other established technologies. Given that most synthetic biology institutions are still at their preliminary phases in terms of commercialization, it is critical that their product is widely accepted and acknowledged by all disciplines that are impacted or will be potentially exposed to the technology.
In the future we hope to take our research and methodologies and present them to the stakeholders discussed within the analysis. Our next steps are to identify institutions and facilities that would benefit from the use of the knowledge transfer strategy and develop sample activities or educational materials that can be used by these facilities to promote the Staphiscope.

References

A.D.A.M Health Solutions. "Methicillin-resistant Staphylococcus Aureus; Community-acquired MRSA (CA-MRSA); Hospital-acquired MRSA (HA-MRSA)." National Center for Biotechnology Information. 9 June 2011. Web. .
"Ethical Issues in Synthetic Biology." Synthetic Biology Project. Web. .
Gaisser, Sibylle, Thomas Reiss, Astrid Lunkes, Kristian Muller, and Hubert Bernauer. "Making the Most of Synthetic Biology : Article : EMBO Reports." Nature Publishing Group : Science Journals, Jobs, and Information. Nature Publishing Group, 2009. Web. .
Herrera, Stephen. "Preparing the World for Synthetic Biology." Technology Review. Jan. 2005. Web. .
The Lancet. "Community-acquired MRSA." 14 Oct. 2006. Web. .
Mitton, Craig, Carol Adair, Emily McKenzie, Scott Patten, and Brenda Waye Perry. "Knowledge Transfer and Exchange: Review and Synthesis of the Literature." University of British Columbia Okanagan and Child and Family Research Institute of BC; University of Calgary; Alberta Mental Health Board. Web. .
Pombo, David. "Community Acquired MRSA." 29 Sept. 2006. Web. .
“Safety and Ethics of Synthetic Life." Organisation for International Dialogue and Conflict Management (Austria). Web. .

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 2011

The following entries pertain to the Quantification Project

Tuesday, May 31, 2011

Transformation of BBa_I20260 from iGem Kit Plate 2, Well 17F.

Wednesday, June 1, 20111

Created a frozen stock of I20260

Inoculated BBa_I0500 and BBa_E0240 from frozen stock already made from last year

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