Team:Waterloo

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                 <h1 style="margin:20px;font-weight:bold"><i>In Vivo</i> Protein Fusion Assembly Using Self Excising Ribozyme</h1>
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                 <h1 style="margin:20px;font-weight:bold"><i>In Vivo</i> Protein Fusion Assembly Using Self Excising Ribozymes</h1>
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<h3 style="color:#0077be">Project</h3>
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<font size="+3"><b>The Intron Project</b></font><br>
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<br>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.</br>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font size="+2">with Mathematical Modelling</font></p>
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<br><br><br>
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<b>1.0 Abstract</b><br><br>
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The Group 1 introns are a widely spread group of ribozymes.  Many of them exhibit self-excising activity. These could be used to generate in vivo recombination libraries by incorporating them with a Cre-lox system, which can facilitate chromosomal gene transposition.  Below we outline a proof-of-concept experiment to display the feasibility of this experiment.<br><br><br>
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<b>2.0 Introduction </b><br><br>
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<b>2.1 Cre-lox system</b><br><br>
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The Cre-lox system is an in-vivo recombination system developed from P1 bacteriophage that is perhaps most familiar to students as a method of gene self-excision (1). The Cre enzyme is a site specific recombinase.  It recognises loxP sites in DNA (34bp), and will either excise, invert or translocate DNA between them (Fig 1).  The Cre enzyme may be designed for expression under specific conditions (eg. IPTG induction), allowing some control over in vivo recombination.  The caveat to using any recombination system to generate fusion proteins in vivo, is that translation of the leftover recombination site sequence or 'scar' can shift codon readings, and/or disrupt protein folding (Fig 2) We propose, therefore, to flank a recombination site, such as the loxP sequence, with a self-excising ribozyme sequence, to create 'scarless' fusion proteins (Fig 2).<br> <br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/3/36/Fig_1.png" alt="Fig 1",width=60%,height=60%,align="absmiddle"><br><center><font size="-1"><b>Fig 1. Schematic of Cre-lox recombination (from (2))</b></font></center></img></p><br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/c/c5/Fig_2.png" alt="Fig 2",width=90%,height=90%,align="absmiddle"><br><font size="-1"><b>Fig 2. Schematic of recombination products a) with a remaining 'scar' and b) with the scar removed from between two fusion protein segments, P<sub>L</sub> and P<sub>R</sub>.  Failure to remove the lox scar may result in disrupted protein folding or mistranslation of the second half of the protein, P<sub>R</sub></b></font></img></p>
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<br> <br> <br>
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<b>2.2 Group I Introns</b><br><br>
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Self-excising ribozymes are an ancient class of introns that can remove themselves from RNA constructs, including mRNA and tRNA (3). The Group I introns are a subset of these self-excising ribozymes, and are widely distributed through simple eukaryotes, fungi, mitochondria, chloroplasts, bacteria and bacteriophage (3).  Group I introns are not yet known to provide a specific biological function, save to excise themselves from important RNA sequences, and thus prevent host death.  One hypothesis is that they are "selfish" remnants of an RNA world (4).  Some Group I introns, for example, code for DNA endonucleases that help them migrate to new sites (3).<br><br>
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The Group I excision mechanism relies on a reactive 'core' rich in RNA secondary structure that conducts two successive transesterifications (5).  Some introns require proteins to help stabilise them, while others are completely independent (5).  The first transesterification involves a nucleophilic attack on the 5' end of the intron, by the 3' OH of a guanosine nucleotide (5) (Fig 3).  Structural rearrangements bring the the 3' end of the exon into proximity with the 3' end of the intron, allowing the second transesterification to occur (5) (Fig 3). The intron is usually then degraded by the host, while the resulting mRNA is translated.<br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/9/95/Fig_3.png" alt="Fig 3",width="390",height="300",align="middle"><br><b>Fig 3. Mechanism of intron self-excision (adapted from (5)) – The construct used above is our experimental construct, further discussed below.  (a) The first transesterification occurs when a GMP attacks the 5' end of the intron (InL-lox-InR). (b) The second transesterification occurs at the 3' end of the intron, led by the 3' end of the exon.  This results resulting in (a) the fusion mRNA for transcription and (d) the intron sequence, to be degraded.</b></img></p>
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<b>2.3 Staphylococcus phage twort ORF142</b><br> <br>
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The Staphylococcus aureus bacteriophage Twort is notable for having three introns within a single gene (6). This ORF142 is a putative structural protein, though its role has not yet been confirmed.  Sequence analysis reveals three highly similar introns, I1, I2 and I3, closely interspersed in the gene.  Experiments show that they are self-excising, and can produce variable splice products, depending on the intron(s) removed (6). This is likely due to their terminal sequence similarity (6).  We have chosen to work with a modified version of these introns due to their independent, self-excision capability, and the low likelihood of disrupting important secondary structure on incorporation of additional RNA.<br> <br>
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2.4 Application <br><br>
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Gene shuffling has been used to create fusion protein libraries of compounds that are more effective than the parent proteins.  Examples include interferon, antibodies and Cry proteins, a family of biological insecticides (7).  Many of these recombination systems, however, rely on in vitro recombination, cloning, then expression and screening of the protein products.  An in vivo recombination system allows the recombination and expression to happen in the same step.  <br><br>
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3.0 Lab and Design<br><br>
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Our project, which continues from last year, is a proof-of-concept for the use of Group I introns in providing scarless removal of an internal sequence.  The marker gene we are using is GFP.  Fig 3 (above) and Fig 7 (below) describe the desired, final construct.  Essentially, successful self-excision of the intron should result in a normal, functional GFP protein.  The final construct will be assembled by joining five "pieces": the two halves of the GFP protein, the two halves of the intron, and a lox recombination site.<br><br>
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<br><h3 style="color:#0077be">1.0 INTRODUCTION</h3>
 
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This design provides a reasonable basis to implement in vivo applications involving RNA level regulatory sequences. The fusion proteins produced surpass strictly what is coded in the DNA. As a result of incorporating ribozyme segments in between two halves of the protein coded in the DNA construct, a regulatory sequence (such as a recombination site) could be included. Since recombination sites can interrupt the functional production of a protein if translated fully (resulting in excess amino acids in the polypeptide), the incorporated ribozyme portions remove them before the translation phase of gene expression so that a functional protein is produced. For example, Cry proteins, which account for the insecticidal activity (toxicity) of <i>Bacillus thuringiensis</i>, could be the fusion protein produced for a particular insecticide. Using our experimental design, the sequence containing the code for the Cry protein (at the DNA stage) is split by ribozyme segments containing a recombination site. In this case, the recombination site is the regulatory sequence that will be removed once transcribed into RNA. At the DNA level, recombination (shuffling) will occur, exchanging DNA strand segments. Therefore, when the shuffled DNA sequence is transcribed into RNA, the recombination site is spliced out of the sequence with the ribozymes, and the resulting RNA code is different than that of the un-shuffled code. Consequently, the translated Cry protein is different. This system would oppose pesticide resistance among the target organism.
 
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<br><h4 style="color:#0077be">1.1 A Little Bit About Group 1 Introns</h4>
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3.1 Mathematical model<br><br>
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<p>
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In order to characterize the relative efficiency of the intron system, a model was developed based on the idea proposed by Kelly et al (8) of comparing steady state fluorescence to a common standard. Our model develops a ratio of protein expression in a system with the intron (P<sub/>intron</sub>) (Fig 4), to expression in a system without the intron (P<sub>GFP</sub>) (Fig 5). It assumes that all reaction rates are linear, and that they remain constant between cell populations. If the rate of mRNA degradation (δ<sub/>M</sub>) is known or estimated, one can approximate the rates of successful mRNA splicing (αS) and failed splicing or folding (α<sub>F</sub>). The resulting measure of efficiency of splicing, then, is α<sub/>S</sub> / (α<sub/>S</sub> + α<sub>F</sub>)  <br><br>
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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 <i>Staphylococcus</i> phage twort.ORF143.
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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.
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<br><h4 style="color:#0077be">1.2 Trans-Esterification Reactions</h4>
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<img src="https://static.igem.org/mediawiki/2012/f/f1/Fig_4.png" alt="Fig 4"><br><b>Fig 4. Model predicting amount of protein formed from a construct containing an intron</b></img><br><br>
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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.</br>
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<br/><div style="width: 350 px; font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/e/e7/Transester.png" width = "350" height "350" style="padding-bottom:0.5em;" /><br/><i>Retreived June 21, 2011 from Self-Splicing RNAs</i> [1] This diagram shows the trans-esterification reaction and splicing of group I introns from a sequence.</div>
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<img src="https://static.igem.org/mediawiki/2012/f/f0/Fig_5.png" alt="Fig 5"><br><b>Fig 5. Model predicting amount of protein formed from an intron-less construct</b></img><br><br>
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<br><h4 style="color:#0077be">1.3 Fusion Proteins</h4>
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<img src="https://static.igem.org/mediawiki/2012/8/85/Fig_6.png" alt="Fig 6"><br><b>Fig 6. The ratio of the rate of protein synthesis with and without an intron</b></img><br><br><br>
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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.
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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. </br>
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3.2 Procedure outline<br><br>
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<br><h4 style="color:#0077be">1.4 The Cre-Lox System</h4>
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We have designed the construct to be built using the meta format of RFC53 in pSB1C3 and pSB1C5. RFC53 is useful for creating scarless recombination between DNA sites using the REN EarI. This cleaves a set distance downstream of their recognition site.<br><br>
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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).</br>
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First, the pieces must be subcloned from pUC57 into pSB1C3 or pSB1C5, which contain the meta-suffix format of RFC53.  The planned construct maps are shown in Fig 7.<br><br>
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<img src="https://static.igem.org/mediawiki/2012/f/fc/Fig_7.png" alt="Fig 7", width=80%,height=80%, align="middle"><br><b>Fig 7. Planned constructs (a) main experimental construct (K576011), (b) positive control (K576013), (c) negative control (K576012) in pSB1C3</b></img><br><br><br>
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<br><h3 style="color:#0077be">2.0 PROJECT IN DETAILS</h3>
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Last year, a diagnostic of the final ligation products revealed unexpected banding. This year, we have re-done the subcloning into pSB1C3, and have proceeded to attempt the ligation of the final construct with more caution.  Previously, diagnostic gels were run only insofar as gel extraction had to be done.  As such there was no confirmation of the ligation pieces before joining them.  This was in part due to the size of the fragments, as several of them are quite small, making it difficult sometimes to visualize them on a gel. <br><br>
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3.3 Lab work, results and conclusions<br><br>
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This year, in addition to conduct the expected laboratory research on the intron project, we invited 20 undergraduate students from the University of Waterloo to join us, in order to teach them some basic molecular biology techniques. Volunteers learned simple molecular biology techniques such as restriction enzyme mapping, miniprep plasmid samples, vector cloning, heat-shock transformation and agarose gel extraction.  <br><br>
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<br><h4 style="color:#0077be">2.1 EXPERIMENTAL DESIGN</h4>    
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The original parts received by our team include left intron (aka Intron 1), right intron (Intron 2), left GFP (GFP 1), right GFP (GFP 2) and a lox region of 34 bp in length. All parts were cloned into pUC57 vector backbone, which is ampicillin resistant. In order to conduct the RFC 53 protocol in our cloning procedure, we need to first sub-clone the R-Intron, L-Intron, L-GFP and R-GFP into pSB1C3 backbone (Chloramphenicol resistant or Cm<sup>R</sup>). <br><br>
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To complete the sub-cloning process, we followed a flow-chart as shown in Figure 8(a)<br><br>
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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.
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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:
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<br/><div style="width: 350 px; font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/1/18/Figure2UWigem2011.png" width = "700" height "250" style="padding-bottom:0.5em;" /><br/><b>Figure 2</b> shows the experimental design of the sequence immediately following transcription. It contains a constituent promoter, RBS Ribosome Binding Site), GFP1, IN1, in-frame stop codon, IN2, GFP2 and TT. The dotted lines and scissors indicate that the introns will be spliced out of the sequence at these points, however, the introns are self-excising.</div><br/>
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<img src="https://static.igem.org/mediawiki/2012/2/2d/Fig_8a.jpg" alt="Fig 8a",width=40%,height=40%, align="middle"><br><right><b>Fig 8a</b></right></img><br><br>
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<br/><div style="width: 350 px; font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/thumb/b/bc/Figure2.png/500px-Figure2.png" width = "600" height "350" style="padding-bottom:0.5em;" /><br/><b>Figure 3</b> is a representative view of the sequence folding in order to catalyze the trans-esterification reaction, however, there are many hairpin loops actually formed. This is the process of post-transcriptional modification. Specifically, Group I intron splicing events utilize a guanosine nucleotide to bind another sequence and dislodge the 5' site, then the cleavage initializes another splicing event with the remaining hydroxyl end to dislodge the rest of the RNA sequence and ligate the remaining exons. The remaining fusion protein code is different than that of the primary transcript.</div><br/>
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We used REN EcoR1 and Pst1 to double digest the original parts and then cloned into pSB1C3 vector. We transformed the cloned vector + insert samples into competent DH5α strain of Escherichia coli and then plated the colonies onto plates with Chloramphenicol.<br><br>
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<br/><div style="width: 350 px; font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/8/89/Posctrlwat.png" width = "600" height "350" style="padding-bottom:0.5em;" /><br/><b>Figure 4</b> shows a non-disruptive ligation scar and active GFP after the self-excision of IN1 and IN2. This is the modified RNA transcript prior to translation..</div><br/>
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Controls are important for plasmid cloning; in addition to our transformation plates, we included vector+ligase control, vector-only control, transformation control and negative (no plasmid) control to check for possibility of contamination and improper techniques. As expected, the transformation control, vector+ligase and vector-only controls had only pink colonies growing on them. The negative control plates were clean. <br><br>
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<br><h4 style="color:#0077be">2.2 CONSTRUCTION MAPs AND RFC 53</h4>
 
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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 and decrease replication rate compared to non-plasmid carriers.
 
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<br/><h5 style="color:#0077be"> 2.2.1 General Construction Map</h5>
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Our transformation plates showed a mix of pink and white colonies; the pink colonies represent the presence of Red fluorescent Protein in pSB1C3. As the multiple cloning site for pSB1C3 lies inside a red fluorescent protein (RFP) gene, successful transformants are white due to disruption of the RFP. We screened 8 of these colonies (2 colonies for each type of insert) and checked the size of the inserts by running an agarose gel, as shown in Figure 8 (b). In addition, we also loaded the digested pUC57 samples to ensure the inserts look identical. The ladder we used in this agarose gel is Fermentas GeneRuler 1kb DNA ladder. We have saved 2 copies of each of 8 clones in our lab freezer and we have also sent one of each type of clone (a total of 4 clones) to the IGEM registry.<br><br>
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<b>Figure 5</b> graphically shows the laboratory procedure for the experimental design in the form of an enzyme map:
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<img src="https://static.igem.org/mediawiki/2012/9/97/Fig_8b.png" alt="Fig 8b"><br><center><b>Fig 8b: Sub-cloning results with Fermentas GeneRuler 1kb DNA ladder</b></center></img><br><br>
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<br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/b/b2/Enzyme_map1.jpg"></img></div></br>
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After successfully cloning the inserts into pSB1C3, we proceed with the RFC 53 protocol to make the main construct. Unfortunately, we were unable to find a clone with the right-sized insert for the L-intron and L-GFP ligation due to time constraints. As a result, we were unable to finish the making of the main construct. <br><br>
-
<li><b>K576005</b> contains the first component of GFP (GFP1)</li>
+
The positive control construct is assembled using RFC 53. Fluorescence is expected to demonstrate that the one amino acid scar from RFC 53 does not disrupt GFP. The negative control construct is assembled using RFC 10. Fluorescence is not expected, in order to demonstrate that presence of the lox site disrupts GFP folding and activity.  <br><br>
-
<li><b>K576003</b> contains the first part of the intron sequence (IN1</li>
+
-
<li><b>J61046</b> contains the lox site</li>
+
-
<li><b>K576006</b> contains the second component of GFP (GFP2)</li>
+
-
<li><b>K576004</b> contains the second part of the intron sequence (IN2)</li>
+
-
<li><b>K576007</b> contains GFP1 and IN1</li>
+
-
<li><b>K576009</b> contains GFP1, IN1 and lox1</li>
+
-
<li><b>K576011</b> contains the promoter (P), ribosomal binding sit (RBS), GFP1, IN1, lox site, IN2, GFP2 and transcriptional terminator (TT). This is the final construct (experimental design)</li>
+
-
+
<img src="https://static.igem.org/mediawiki/2012/4/40/Fig_8c.png" alt="Fig 8c"><br><center><b>Fig 8(c): image of the gel used to extract fragments for the control constructs</b></center></img><br><br>
-
<br><h5 style="color:#0077be">2.1.2 Controls' Construction Map</h5>
+
-
+
-
<br><p>
+
-
<p>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.</p>
+
-
<br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/thumb/c/c4/Figure5a.png/430px-Figure5a.png" ></img></br>
+
Figure 8(c) is an example of a gel used to prepare gel extracted fragments for the two control constructs. We were unable to get any successfully transformed colonies for either construct.<br><br>  
-
<b>Figure 6</b> represents the positive control. For consistency, GFP1 and GFP2 are separated by RFC53 (RFC25 could have also be used), which should not disrupt expression of a functional GFP.</div><br>
+
-
<br><p>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 7 shown below details the negative control design:</p></br>
+
Two criticisms in last year's experimental design have been highlighted this year, which may explain some of the difficulties in creating a working construct.  First, many of the fragments being used are relatively small, which makes it difficult to isolate them for ligation.  A suggested solution is to use PCR to amplify or introduce, for example, the 34bp lox site to the construct. Second, some of the REN being used for double digests are very close to each other on the plasmid (< 10bp). This may be impeding the RENs' ability to bind and cleave DNA.
 +
An alternative design to the one proposed here would be to create a gene with multiple intron sites to allow continuous mRNA recombination in vivo, rather than a one time recombination event at the DNA level (6).<br><br><br><br>
-
<br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/thumb/2/2a/Figure4.png/560px-Figure4.png"></img></br>
+
3.4 Side project: Promoter expression quantification<br><br>
-
<b>Figure 7</b> is an illustration of the negative control containing the interrupted GFP with an in-frame stop codon, ensuring that it is not expressed. Since this sequence contains no excision sites (Group I introns), we do not expect any modification to this area of the RNA.</div><br>
+
In parallel with the lab work of constructing the intron system, progress was made at our lab towards implementing a framework for measuring relative fluorescence of cell cultures, using the protocol described by Kelly et al. (8). Such measurements are necessary in order to quantify the notion of intron efficiency developed in our model.<br><br>
-
<p><br>The figure below shows the construction map for the controls.</br>
+
Work on this project is ongoing. The steps taken this summer were:<br>
-
<br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/0/08/Enzyme_map3.jpg"></img></div></br>
+
&nbsp;&nbsp;• identification and acquisition of appropriate materials and equipment access<br>  
 +
<!-- <ul> not working!!!!-->
 +
&nbsp;&nbsp;• training of four undergraduate students, separate from the main lab project, in essential lab techniques<br>
 +
&nbsp;&nbsp;• test-running the protocol and troubleshooting errors.<br><br>
 +
Once a quantification system is successfully implemented, our team will be also be equipped to begin making measurements of many other registry parts, according to the RPU standard proposed by Kelly et al. (8). Our vision is for RPU quantification of existing registry parts to be a standard component of UW iGEM's yearly activities, beginning with constitutive promoters and expanding into measurements of more complex devices.<br><br><br><br><br>
-
<li><b>K576005</b> contains the first component of GFP (GFP1)</li>
+
<b>References</b><br>
-
<li><b>K576006</b> contains the second component of GFP (GFP2)</li>
+
(1) Nagy, A. (2000). Cre Recombinase: The Universal Reagent for Genome Tailoring. Genesis, 26(2), 99–109.<br>
-
<li><b>K576013</b> contains the promoter (P), ribosomal binding site (RBS), GFP and transcriptional terminator (TT). This is the positive control.</li>
+
(2) The Jackson Laboratory. (n.d.). Introduction to Cre-lox technology. (<a href="http://cre.jax.org/introduction.html">http://cre.jax.org/introduction.html</a>)<br>
-
<li><b>K576005</b> contains the first component of GFP (GFP1))</li>
+
(3) Haugen, P., Simon, D. M., & Bhattacharya, D. (2005). The natural history of group I introns. Trends in Genetics, 21(2), 111–119. <br>
-
<li><b>J61046</b> contains the lox site</li>
+
(4) Lambowitz, A. M., Caprara, M. G., Zimmerly, S., & Perlman, P. S. (1999). The RNA World, Second Edition.  New York: Cold Spring Harbor Laboratory Press, 451–486.<br>
-
<li><b>K576006</b> contains the second component of GFP (GFP2)</li>
+
(5) Cech, T. R. (1990). Self-Splicing of Group I Introns. Annual Reviews in Biochemistry, 59, 543 – 568. <br>
-
<li><b><K576012></b> contains the promoter (P), ribosomal binding site (RBS), GFP1, lox site, GFP2 and transcriptional terminator (TT). This is the negative control.</li>
+
(6) Landthaler, M., & Shub, D. A. (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. Proceedings of the National Academy of Sciences of the United States of America, 96(12), 7005–7010.<br>
-
</p>
+
(7) Glick, B. R., Pasternak, J. J., & Patten, C. L. (2010). Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition.  Washington DC: ASM Press.<br>
 +
(8) Kelly, J. R., Rubin, A. J., Davis, J. H., Ajo-Franklin, C. M., Cumbers, J., Czar, M. J., de Mora, K., et al. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3:4.<br><br>
-
</p>
 
-
 
-
<br><h3  style="color:#0077be">2.3 MAKING THE CONSTRUCT WITH RFC 53</h3>
 
-
 
-
<p><br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/thumb/2/2f/53.png/500px-53.png"></img></br>
 
-
<b>Figure 9:</b> Making the Construct: Parts were sequenced into PUC57, digested and ligated into PSB1C3.</div><br />
 
-
<b>Figure 9</b> is a flow chart of the general work flow involved in the construction of our experimental plasmid, as per RFC53 conventions.<br><br>
 
-
<ol>
 
-
<li>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.</li>
 
-
<li>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.</li>
 
-
<li>3) The two components (insert and vector) are ligated together to produce the final construct.</li>
 
-
<li>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.</li>
 
-
</ol>
 
-
</p>
 
-
 
-
<br><h3 style="color:#0077be">2.3 Preliminary Testing</h3>
 
-
 
-
<br><p>
 
-
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.
 
-
 
-
<p><br><div style="font-size:90%; text-align:center;"><img src="https://static.igem.org/mediawiki/2011/a/a5/Figure10UWigem2011.png"></img></br>
 
-
<b>Figure 10:</b> shows the diagnostic digestion from September 16, 2011: All subclones shown to be correct, however, positive control not correct. Relative placement of GFPs and INTS required further verification and showed incorrect placement. This accounts for lack of fluorescence in final stage.</div><br>
 
-
<br><p>
 
-
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.
 
-
</p>   
 
-
 
 
-
<br><h3 style="color:#0077be">3.0 PRACTICAL APPLICATION </h3>
 
-
 
-
<br><p>
 
-
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.
 
-
<br><br />
 
-
<h3 style="color:#0077be">4.0 REFERENCES</h3><br />
 
-
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.
 
-
<br /><br />
 
-
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.
 
-
<br /><br />
 
-
Bernstein, K.E., Bunting, M., Capecchi, M.R., Greer, J.M., Thomas, K.R. (1999). Targeting genes for self-excision in the germ line.
 
-
<br /><br />
 
-
Cassin, P., Gambier, R., Scheppler, J. (2000). Biotechnology Explorations: Applying the Fundamentals. Washington, DC: ASM Press.
 
-
<br /><br />
 
-
Cech, T. (1990). Self-Splicing of Group I Introns. Biochemistry 59:543-8.
 
-
<br /><br />
 
-
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).
 
-
<br /><br />
 
-
Glick, B., Pasternak, J., Pattern, C. (2010). Molecular Biotechnology Principles and Applications of Recombinant DNA Fourth Edition. Washington, DC: ASM Press.
 
-
<br /><br />
 
-
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.
 
-
<br /><br />
 
-
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.
 
-
<br /><br />
 
-
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.
 
-
<br /><br />
 
-
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.
 
-
<br /><br />
 
-
Minnick, M.F., Raghavan, R. (2009). Group I Introns and Inteins: Disparate Origins but Convergent Parasitic Strategies. Journal of Bacteriology. 191 (20), 6193-6202.
 
-
<br /><br />
 
-
Peters Ph.D., Pamela (N/A). Restriction Enzymes Background Paper An Excellence Classic Collection. (http://www.accessexcellence.org/AE/AEC/CC/restriction.php).
 
-
<br /><br />
 
-
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
 
-
<br /><br />
 
                 </div>
                 </div>
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<div class="innerContent">
<div class="innerContent">
<h2 style="color:#0077be">UW iGEM OUTREACH PROJECTS 2011-12</h2><br />
<h2 style="color:#0077be">UW iGEM OUTREACH PROJECTS 2011-12</h2><br />
-
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 we aim to provide the baseline knowledge required that will allow our community members to form a fact-based opinion.<br/><br/>
+
<br>Our Mission: Raise awareness on issues concerning synthetic biology to make informed, fact-based opinions.</br>
 +
<br><b>Introduction</b></br>
 +
<br>This year’s UW iGEM’s Outreach sub-team has learnt a lot from its experiences. We were met with a few hurdles with regards to planning and implementing the myriad of events we had hoped to have to engage our community. Though with every step there was much to be learnt and we have decided to discuss all the great events we did have planned and are in the process of planning. Along with that we thought it would be a great resource to create a guide that discussed various different types of outreach events you can hold and what hurdles you could potentially face (as we did) in terms of implementation. </br>
 +
<br><b>Successes</b></br>
 +
Despite the barriers that we faced, we still had numerous successes. We were still able to develop Community Bricks for most of our programs so other Outreach members can implement them as well. The <b>Community Bricks</b> we were able to post include:
 +
<ol>
 +
<li> 1. Grade 11 Synthetic Biology Workshop Program with <a href="http://bit.ly/VqhuSW"> Interactive Activities</a></li>
 +
<li> 2. <a href="http://bit.ly/SM8js3">DIY, At Home Gel Electrophoresis</a></li>
 +
<li> 3. <a href="http://bit.ly/QveKQQ">Basic Lab Training Workshops</a> </li>
 +
<li> 4. <a href="http://bit.ly/R8kGlc">Communications Plan Template</a> and Example for Your Large-Scale Outreach Programs </li>
 +
<li> 5. <a href="http://bit.ly/P7tyF6">Tips and Tricks to Have a Successful Outreach Event</a></li>
-
This year, UW iGEM: Outreach focused on designing and running workshops targeted at schoolchildren. We hoped to share our love and passion for biology with tomorrow's future scientists and engineers. We plan to continue building on what we currently have and to eventually develop a complete syllabus for all grade levels. These workshops will be available for download for other educators and enthusiasts interested in their own outreach.<br/><br/>
+
</ol>
 +
<br>As well as…<br>
 +
<a href="http://sns.vroc.ca/p.jsp?i=17">Educational Video Podcast</a> featuring the iGEM Outreach Lead Ekta Bibra on Synthetic Biology for Virtual Researcher On Call (organization providing educational resources to students and teachers through online web conferences or podcasts in Ontario, Canada). This material will be openly available for students and teachers to view and learn from across the province. This will be available for you to view at the regionals this year!
 +
<br>As well, we are working on a core social media campaign in which we amplify awareness with minimal costs, gain a larger audience base to ease overall promotion and easily apply Outreach’s over-arching goal of raising awareness on synthetic biology. Make sure you follow us and our progressions on: </br>
 +
<br>Twitter: @Waterloo_iGEM</br>
 +
<br>Facebook: Waterloo iGEM page</br>
 +
<b><br>Outreach Activities</b></br>
 +
<br>The divisions of our outreach events are regularly divided into seasonal timings, from Fall, Winter to Spring. This year these were the events that we had initially planned on implementing:</br>
 +
<br>
 +
<ol>
 +
<b><li> BioTalks Open Panel Discussion and Lecture Series</br></b>
 +
<a href="http://bit.ly/R8kGlc">Community Brick (Communications Plan Plus Example)</a><br />
 +
Purpose: Develop an exciting discussion forum between science undergraduates and industry specialists within biotechnology to encourage science entrepreneurship, innovation and industrial advice.<br />
 +
How: Organize a large-scale open panel discussion involving brief 20 minute talks from key industry leaders followed by a question and answer period that can supplement what has been demonstrated within the short lectures.<br />
 +
Type: Education/Awareness </li><br />
 +
<br><b><li> Engineering Science Quest (ESQ)</br></b>
 +
<a href="http://bit.ly/SM8js3"> View Community Brick</a><br />
 +
Purpose: A tradition for the UW iGEM Outreach team to carry out, this workshop is held by ESQ to develop hands-on learning activities and workshops for students from Grade 3-12 to promote science/engineering education and outreach.<br />
 +
How: Development of a do-it-yourself workshop for Grade 12 students that incorporates science into their everyday lives. Also created a Community Brick.<br />
 +
Type: Education</li><br />
 +
<br><b><li>Hot Chocolate Event</br></b>
 +
Purpose: To spread the word on campus of UWaterloo iGEM, gain presence amongst students of who we are and promote our social media accounts on Twitter and FaceBook.<br />
 +
How: Selling hot chocolate for 50 cents along with a small paper tag of our FaceBook and Twitter page. <br />
 +
Type: Marketing/Promotion</li><br />
 +
<br><b><li>Movie Night</br></b>
 +
Purpose: In a social setting, show what types of content regarding biotechnology and social media are presented to the public. Demonstrate how that affects the overall perspective that they have towards the topic and how it shapes their future opinions.<br />
 +
How: Show a very popular movie that has recently played or a well-known classic to the students at the University. <br />
 +
Type: Awareness</li><br />
 +
<br><li><b>Basic Lab Training </b><br />
 +
<a href="http://bit.ly/QveKQQ"> Community Brick</a><br />
 +
Purpose: Since we are a co-operative education school (meaning many students within our University do 5 or more 4 month internships with employers) it helps to have a kick-start on basic lab skills and techniques to add onto their resumes when they may not have that experience yet. This will help to make them more competitive when applying for positions. <br />
 +
How: Hold a one day training session where University students are taught basic skills used everyday in our wet lab.<br />
 +
Type: Education</li><br />
 +
<br><li><b>Grade 11 Synthetic Biology Workshop </b><br />
 +
<a href="http://bit.ly/VqhuSW"> Community Brick</a><br />
 +
Purpose : Similar in nature to UW iGEM 2011 Outreach’s Grade 12 Synthetic Biology Workshop (you can find the Community Brick for this, this interactive workshop’s aim was to promote open discussion on industries and current issues within synthetic biology.<br />
 +
How: Hold a workshop using lab space and the development of two new activities that would suit the Grade 11 Biology curriculum.<br />
 +
Type: Education</li><br />
 +
<br><li><b> Tips and Tricks to Have a Successful Outreach Event</b><br />
 +
<a href="http://bit.ly/P7tyF6"> Community Brick</a><br />
 +
Purpose : To take what has been learnt throughout the year and streamline the key values into a valuable guide for those interested in pursuing outreach activities. <br />
 +
How: Develop a brief information sheet that puts together key points for organizing outreach events.<br />
 +
Type: Educational</li><br />
 +
</ol>
-
<h3 style="color:#0077be">Workshop Materials</h3><br/>
+
<br><b>IN DEPTH DESCRIPTION OF EVENTS:</b><br />
-
<br/>
+
<br><ol><li><b>BIOTALKS OPEN PANEL DISCUSSION</b><br />
-
We have submitted two community bricks! One for our Grade 12 workshop and the other for our Engineering Science Quest activity for Grades 3-4. The downloadable material is the same as what you can find here on our wiki page. <br/>
+
<a href="http://bit.ly/R8kGlc"> Community Brick (Communications Plan Plus Example)</a><br /><br />
-
            -Synthetic Biology and You: <a href="http://openwetware.org/wiki/IGEM_Outreach:Synthetic_Biology_and_You_Interactive_Workshop_%28Gr._11-12%29">Interactive Workshop for Grades 11-12</a><br/>
+
This event we are planning will be more than just a lecture series, but it is meant to be so much more. It is an experience, a place of discussion and intellectual exchange and of learning. Those are some of the visions we have for this event.<br /><br />
-
            -All About Bacteria: <a href="http://openwetware.org/wiki/IGEM_Outreach:All_About_Bacteria:_How_Clean_Are_Your_Hands%3F">How Clean Are Your Hands?</a><br>
+
By having key industry speakers do brief 20 minute lectures on topics such as: Clinical Trial Drug Development, Commercialization of Biomass and Energy Products and Entrepreneurial Barriers for Biotechnological Companies, we are hoping to bridge the level of discomfort scientists have when it comes to business. We want to encourage innovation amongst the highly technical individuals within our university and provide an open forum of educational resources for them to gain knowledge.<br /><br />
 +
Currently, UW iGEM has a core team of members working on this initiative and we are hoping to implement the BioTalks event in March 2013. We have completed our communications plan, gathered 3 speakers from the Pharmaceutical, Agricultural and Renewable Energy sectors and have separated the organizational team into three components: Director and Lead of Social Media Marketing (Ekta Bibra), Internal Communications and Logistics Lead (Anjali Arya) and Vision and Strategy  Lead (Angela Biskupovic). Now we are working on meeting the appropriate potential sponsors and advisors.<br /><br />
 +
<b>The creation of a communications plan is very helpful in creating clear-cut objectives and values, giving your team an over-arching mission and have stakeholders who you are proposing this event to understand exactly what you are trying to do. We have created a Community Brick that demonstrates a template for writing a Communications Plan as well as our BioTalks example that we are currently proposing to potential sponsors and advisors.<br /><br /></b>
 +
Our next steps involve, the development of an aggressive marketing strategy through social media and direct marketing, raising funds and all the logistical work involved with a large-scale event such as this. Please stay tuned! If you have questions on this event feel free to contact us and we would love to share ideas with you!<br /><br />
 +
Contact the BioTalks team at: uwigem.outreach.hp@gmail.com (Ekta Bibra, Outreach Lead)<br /></li>
-
<br/>
+
<br><li><b>ENGINEERING SCIENCE QUEST (ESQ)<br /></li></b>
-
<strong>Grades 3-4: All About Bacteria</strong> - <a href="https://static.igem.org/mediawiki/2011/3/36/Grades_3-4_-_All_About_Bacteria.pdf">Outline</a> | <a href="https://static.igem.org/mediawiki/2011/3/3d/Grades_3-4_-_All_About_Bacteria_%28Handout%29.pdf">Handout</a> <em>(Duration: 2-day workshop, 1.5 hours total)</em><br/>
+
<a href="http://bit.ly/SM8js3"> Community Brick Available for Protocol and Slide Deck</a><br /><br />
 +
Outreach took a part in the Engineering Science Quest at University of Waterloo by holding a lab activity for high school students (ages 14-17). The idea was to show students that it is easy to do Science at home.<br /><br />
 +
Gel Electrophoresis was performed using all supplies available at home (except for agarose- but one can use Jello too). We performed this activity with 3 different groups of ~10 students each time over the span of 2 months. We paired students together and gave them a chance to work in a team and learn some basic lab techniques such as pipetting, understanding the idea of having a medium for the electrons to flow in and much more.<br /><br />
 +
Students were also given a formal presentation at the end of the activity where the theory of Gel Electrophoresis was explained and were at the end asked a few questions for small prizes.<br /><br />
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Students were able to separate food dye (based on their  charge) successfully; as 4 out of 5 groups, on average, did see a proper separation of colours.<br />
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<Strong>Grades 5-6: All About DNA</strong> - <a href="https://static.igem.org/mediawiki/2011/d/da/Grades_5-6_-_All_About_DNA.pdf">Outline</a> <em>(Duration: 1 hour)</em><br/>
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<br><li><b>HOT CHOCOLATE EVENT<br /></li></b></br>
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<br>The purpose of this event was to bring the UW campus community together and educate them about the field of synthetic biology through iGEM.<br />
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<br>The idea was to serve hot chocolate to individuals wishing to buy it for 50 cents a cup. While they are getting their drink served to them, iGEM members will be there engaging in conversations with the various students and faculty, discussing with them the history of synthetic biology and how it has evolved over the years.<br />
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<br>Hosting such an event especially a one that involves serving food to the local public, requires a lot of permission seeking for various personnels.<br />
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<br>The following protocol must be followed at UW in order to serve food publically anywhere on campus:<br />
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<ul><br><li>When serving food publically, one must first contact Feds and let them be aware of your ideas to host such event well in advance (estimated 4 weeks prior). Space is very limited on campus therefore letting Feds know of your plans somewhat secures your spot on campus for that day and time.</li>
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<br><li>Next comes having the Waterloo Health Region approve your plans and your food before your serve them to the public. This is a fairly long process and usually teams/clubs/groups are advised to fill out this form and get in touch with Waterloo Health Region at least 8 weeks prior to your event.</li>
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<br><li>While you are still waiting to hear from the Waterloo Health Region, make sure you keep in touch with what else is going on around campus that week that could possibly interfere with your event. For example I our case, UW’s Welcome Week BBQ lunch was being held at the same time same area, same day. Try and contact those individuals so you can work out the space and equipment (if sharing) so no issues arise on the day off the event.</li>
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<br><li>In addition, have your ingredients (same ones listed on the WHN form) arranged and readily available on the day of your event.</li>
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<br><li>Lastly, to save yourself time, if you will be using electronic equipments or large equipments such as water thermals that require you to bring hot water externally, have it arranged close to your location and already approved by the “company” supplying it to you.</li>
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<br><li>Throughout this whole process keep in mind that communication is key. You will be in touch with various individuals from various groups and so be as descriptive and detailed as possible to get your point across.</li>
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</ul>
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<br><li><b>MOVIE NIGHT/ENTERTAINMENT NIGHT<br /></li></b></br>
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<br>The purpose of this event was to show a movie related to Synthetic Biology in relation to what is new today (for a possible discussion later on).<br />
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<br>When starting an outreach event involving entertainment for the first time one must. Understand that the turnout may not be very high but remember it is a first-time event. Especially if it is held during midterm period.<br />
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<br>Next you must focus on getting in touch with Feds again and communicating with them to try and find a place where not only can you show a movie but also accommodate for the amount of people you expect to turn up for this event.  This can be tricky if you have a very specific date picked out. Always have a range of dates as a backup plan because since space if limited on campus, you might not always get your desired location at the day and time you want it.<br />
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<br>If there are specific individuals in-charge of these events make sure they are aware of not only where to find the appropriate equipment needed to show the movie on campus but also the process involved in the rental. There are very strict protocols that need to be followed in regards to electrical equipment being rented at UW. Get in touch with media doc. for more details for UW.<br />
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<br>Another thing to keep in mind is that University of Waterloo is only allowed to show licensed movies/videos on campus. Feds is usually a great help in this process. They tend to have a huge list of movies available for you to choose from and in most cases the movie you want to show will be included in that licensed list. If not try and get in touch with one of the Feds executives or clubs manager to see if they can assist you in getting a license to show that movie on campus. The movie however will be rented from the closest store.<br />
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<br>One last thing to be kept in mind is, if your group plans on serving food at this event, make sure to follow the steps similar to the ones listed above for the food event.<br />
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<strong>Grade 12: Synthetic Biology and You</strong> - <a href="https://static.igem.org/mediawiki/2011/b/b4/Waterloo_iGem_Outreach_Materials_for_Download.zip">Materials</a> | <a href="https://static.igem.org/mediawiki/2011/c/cb/SynBio%2C_Biotech_and_You_Part_1.zip">Ppt (part 1)</a> | <a href="https://static.igem.org/mediawiki/2011/1/12/SynBio%2C_Biotech_and_You_Part_2.zip">Ppt (part 2)</a> <em>(Duration: 2-3 hours)</em>
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<br><li><b>LABORATORY EVENT</li></b>
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<a href="http://bit.ly/QveKQQ"> Community Brick Available for Protocol </a><br />
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Are you interested in doing any of these activities with kids around this age? Feel free to use any of our materials above and/or 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. And definitely a lot of fun!<br /><br />
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<br>The purpose of this event was to educate science students about the basic and complicated lab techniques that are required during their undergraduate years here at Waterloo.<br />
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<br>This exercise followed a similar pattern to our ESQ exercises, however are made more directed towards first and second year students.<br />
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<br>Since this activity is very hands on, laboratory safety protocol is very important to follow. Each student that wants to participate in this activity must have their WHMIS certification before they show up for the event.<br />
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<br>For activities like this only a lab space can be used to perform the experiments. Therefore it is crucial that you get an instructor’s permission to his/her lab space. With regular lab sections utilizing the lab space for a minimum for 3 hours per section, time is very critical. In most cases this even will have to be held when a specific lab is over for the term.<br />
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<br>Details of the experiments being performed must be discussed by the instructor before anything is agreed upon. These details should include, the time and date of your event, what kind of experiments the students will be performing as well as what kind of equipment will be provided to them and if necessary what kind of equipment is required by the instructor of the lab to provide for us.<br />
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<br>Costs of the equipment and materials being used will also have to be calculated just in case there needs to be a charge placed on this.<br />
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<br>Keep in mind that lab spaces are not entirely meant for a large group of students. Prior to deciding on the exact date and time for your event, try and put out a survey to see how many students are actually interested. This will help you decide whether you should have one or two sessions and whether or not they should be on the same time and date or not.<br />
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<br>Before finalizing the details for the event just prepare a timeline and run it through the instructor as well as the volunteers teaching the students so everyone is on the same page.<br />
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<br>If someone within your team or outside of your team is making a commitment for the event that is critical for its execution, ensure that they either understand that once they make a commitment to a certain time they cannot leave or make them sign a contractual form binding them to the event. This will ensure no last minute cancellations.<br />
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<h3 style="color:#0077be">Events</h3><br/>
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<br><li><b>EXTERNAL WORKSHOPS<br /></li></b>  
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We were also fortunate enough this year to have been given the opportunity to run our workshops at two different outreach events, both of which were on a grand  scale. Now, we'd like to share our experiences with you.<br /><br />
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<a href="http://bit.ly/VqhuSW"> Community Brick (Synthetic Biology and You, Gr.11)</a><br />
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<h3 style="color:#0077be">Grade 12 Outreach Workshop: March 25th, 2011</h3><br />
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<br>The purpose of this event is to education younger generation (Gr. 11 and 12 students) about the field of synthetic biology, what it involves and what kind of future it holds for them. The idea behind it is to let them be aware of the opportunities out there for them in the field of science.<br />
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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. <br /><br />
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<br>This workshop involves a selected science class from the local high school to come to campus for a whole day event. Being able to contact a High School teacher and letting them know about this opportunity for their students is the main part. It is slightly easier for the beginning year to keep it limited to local High Schools just because that way you are limiting any communication and travelling issues. Team members could also get in touch with their old high school science teacher to see if they are interested to attend.<br />
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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.
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<br>If the teacher is hearing about this for the first time it is usually beneficial to have brochures, pamphlets or postcards to distribute to the prospects. This makes sure that they leave with enough information to get them to start thinking about the idea of getting their students to come and learn about synthetic biology.<br />
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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.
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<br>Sometimes getting in touch with teachers can be hard. If you University/College has outreach programs that associate with high schools, get in touch with them, they can be a great aid in terms of networking with the High School teachers.<br />
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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. <br /><br />
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<br>Keep in mind that even if the High School is local, there needs to be a written contest of the teacher agreeing to perform this activity on the given date and any changes made should be informed to the other party in a well advanced notice. This should also be run through the school’s administration to avoid any last minute changes or cancellations. Set one effective deadline and aim to plan everything accordingly.<br />
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  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™! <br /><br />
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<br>In these types of activities communication between you group, the school and the teacher is essential.<br />
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<br>After those details have been finalized, similar room booking directions can be followed as listed before.<br /></ol>
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<div style="text-align: center;"><img src="https://static.igem.org/mediawiki/2011/e/e5/Watout1.png" width="350" height="270"><img src="https://static.igem.org/mediawiki/2011/d/d6/Outreach2.png" width="350" height="270"><br/><br/></div>
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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. <br /><br />
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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.
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  <div style="text-align: center;"><img src="https://static.igem.org/mediawiki/2011/9/98/Watout3.png" width="350" height="270"><img src="https://static.igem.org/mediawiki/2011/7/7c/Watout4.png" width="350" height="270"><br/><br/></div>
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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. <br /><br />
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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!
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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. <br/><br/>
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<h3 style="color:#0077be">Engineering Science Quest: July-August 2011</h3>
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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.<br /><br />
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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.<br /><br />
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  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. <br /><br />
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<div style="text-align: center;"><img src="https://static.igem.org/mediawiki/2011/1/1c/Watout5.png" width="350" height="270"><img src="https://static.igem.org/mediawiki/2011/2/25/Watout6.png" width="350" height="270"><br/><br/></div>
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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.
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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. <br /><br />
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<div style="text-align: center;"><img src="https://static.igem.org/mediawiki/2011/6/6c/Watout7.png" width="350" height="270"><img src="https://static.igem.org/mediawiki/2011/2/2c/Watout8.png" width="350" height="270"><br/><br/></div>
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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.
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<br /><br />
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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.  
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<h2 style="color:#0077be">Human Practice</h2><br>
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<h2 style="color:#0077be">Determining The Factors Which Affect Perception of Synthetic Biology: A Multiple Regression Analysis</h2><br>
Despite synthetic biology's rapidly growing importance in a wide variety of fields including energy and health, it is still relatively unknown to the population at large. While some may have a vague notion of what synthetic biology is and its potential impact, most do not have anything to associate it with. In fact, some may even find the juxtaposition of artificial (synthetic) and natural (biology) confusing or contradictory. We at the iGEM University of Waterloo Human Practices team believe this represents a prime opportunity to help shape the public perception of synthetic biology and allay the fear and paranoia typically associated with the emergence of similar new fields of study. To do this effectively, we believe it's necessary to examine closely what factors or characteristics may affect a person's perception of synthetic biology. The purpose of this study, then, is to use statistical analysis, specifically regression modelling, to quantify these factors and their effect on perception. We created a survey to gather the data necessary for this analysis. It consists of three sections: first, background information to help identify the relevant factors for each respondent; next, a "pop" quiz designed to provide insight into the respondent's knowledge of synthetic biology; finally, a section that relates to the respondent's perception of synthetic biology and its uses.
Despite synthetic biology's rapidly growing importance in a wide variety of fields including energy and health, it is still relatively unknown to the population at large. While some may have a vague notion of what synthetic biology is and its potential impact, most do not have anything to associate it with. In fact, some may even find the juxtaposition of artificial (synthetic) and natural (biology) confusing or contradictory. We at the iGEM University of Waterloo Human Practices team believe this represents a prime opportunity to help shape the public perception of synthetic biology and allay the fear and paranoia typically associated with the emergence of similar new fields of study. To do this effectively, we believe it's necessary to examine closely what factors or characteristics may affect a person's perception of synthetic biology. The purpose of this study, then, is to use statistical analysis, specifically regression modelling, to quantify these factors and their effect on perception. We created a survey to gather the data necessary for this analysis. It consists of three sections: first, background information to help identify the relevant factors for each respondent; next, a "pop" quiz designed to provide insight into the respondent's knowledge of synthetic biology; finally, a section that relates to the respondent's perception of synthetic biology and its uses.
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<div class="innerHeader"><h2>OUR TEAM!</h2></div>
<div class="innerHeader"><h2>OUR TEAM!</h2></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/b/b0/Screen_Shot_2011-09-29_at_12.15.05_AM.png" alt="Peter", width="160" height="200">Peter Hong - Director</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/b/b0/Screen_Shot_2011-09-29_at_12.15.05_AM.png" alt="Peter", width="160" height="200">Peter Hong<br>Director</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2010/e/e6/Teamprofile08_UW.jpg" alt="Ekta"></img><br />Ekta Bibra - Outreach Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/b/b9/T05.jpg" alt="Linda", width="147" height="200"></img><br /> Linda Yang<br>Assistant Director</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/e/e1/AngelaUW2011%28200x160%29.jpg" alt="Angela"></img><br />Angela Biskupovic - Human Practice Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2010/e/e6/Teamprofile08_UW.jpg" alt="Ekta"></img><br />Ekta Bibra<br>Outreach Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/f/fc/T02.jpg" alt="Anjaji", width="149" height="200"></img><br /> Anjali Arya<br>Outreach Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/8/87/Kasiawaterloo.jpg" alt="Kasia", width="160" height="200">Kasia Karpinska-Leydier - Lab Team</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/1/1f/T09.jpg" alt="Urooj", width="147" height="160"></img><br /> Urooj Kishor<br>Outreach Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2010/f/fb/Teamprofile29_UW.jpg" alt="Jordan"></img>Jordan Lapointe - Mathematical Modelling Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/e/e1/AngelaUW2011%28200x160%29.jpg" alt="Angela"></img><br />Angela Biskupovic<br>Human Practices Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/4/4c/T08.jpg" alt="Simon", width="144" height="200"></img><br />Simon Burru<br>Human Practices Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/c/c1/T01.jpg" alt="Aaron", width="240" height="180"></img><br />Aaron Bender<br>Lab Project Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/2/2e/UW2012-T10.jpg" alt="Artus", width="150" height="160"></img><br />Dongbin Zhang<br>Lab Project Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/d/d4/T03.jpeg" alt="Denise"></img><br />Denise Lieuson<br>Lab Project Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/f/fd/T11.jpg" alt="Emily",width="170" height="165"></img><br />Emily JunWen Li<br>Lab Project Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/1/14/T07.JPG" alt="Rummy", width="148" height="202"></img><br />Rummy Chowdhury<br>Lab Project Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2011/8/87/Kasiawaterloo.jpg" alt="Kasia", width="160" height="200">Kasia Karpinska-Leydier<br>Lab Team</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/c/c6/T12.jpg" alt="Ashley", width="180" height="135">Ashley Ross<br>Lab Team</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/d/d0/T13.jpg" alt="Reena", width="150" height="200">Reena Paink<br>Lab Team</img></div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2010/f/fb/Teamprofile29_UW.jpg" alt="Jordan"></img>Jordan Lapointe<br>Mathematical Modelling Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/5/5e/T06.png" alt="Paul",width="140" height="171"></img><br />Paul Reginato<br>Mathematical Modelling Leader</div>
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<div class="teamProfile"><img src="https://static.igem.org/mediawiki/2012/8/89/IGEM_-_Kathryn_Scannell_-_Mathematical_Modelling_Team.JPG" alt="Kathryn",width="140" height="171"></img><br />Kathryn Scannell<br>Mathematical Modelling Team</div>
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<h3>UW's parts for 2011.</h3>
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<h3>UW's parts for 2012</h3>
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_K576003">BBa_K576003</a>  -    RNA      - Left part of self-excising ribozyme<br /><br />
<a href="http://partsregistry.org/Part:BBa_K576003">BBa_K576003</a>  -    RNA      - Left part of self-excising ribozyme<br /><br />
Line 623: Line 627:
<a href="http://partsregistry.org/Part:BBa_K576012">BBa_K576012 </a>  -    Reporter - Negative control of the experiment. The lox recombination site interrupts the GFP expression <br /><br />
<a href="http://partsregistry.org/Part:BBa_K576012">BBa_K576012 </a>  -    Reporter - Negative control of the experiment. The lox recombination site interrupts the GFP expression <br /><br />
<a href="http://partsregistry.org/Part:BBa_K576013">BBa_K576013 </a>  -    Reporter - Positive control of the experiment. Everything in between has been cut out by the self-excising intron and the GFP is fully expressed. <br /><br />
<a href="http://partsregistry.org/Part:BBa_K576013">BBa_K576013 </a>  -    Reporter - Positive control of the experiment. Everything in between has been cut out by the self-excising intron and the GFP is fully expressed. <br /><br />
 +
<HR/>
 +
The UW 2012 lab project is a continuation from the 2011 one, thus no new parts are introduced.<br><br>
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               <div class="aContentMenuOption" onMouseOver="aContentMenuHover(1,true)" onMouseOut="aContentMenuHover(1,false)" onMouseDown="aContentMenuSelect(1)">Lab Work<br>Overview</div>
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               <div class="aContentMenuOption" onMouseOver="aContentMenuHover(2,true)" onMouseOut="aContentMenuHover(2,false)" onMouseDown="aContentMenuSelect(2)">Lab Journal<br>Entries</div>
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-
<h3>Lab Notebook 2011</h3><br/>
 
-
<b>The following entries pertain to the Quantification Project</b><br/><br/>
 
-
<h5>Tuesday, May 31, 2011</h5>
+
<img src="https://static.igem.org/mediawiki/2012/a/a0/UW2012-N01.png" alt="N01"></img><br>
-
<li>Transformation of BBa_I20260 from iGem Kit Plate 2, Well 17F.</li><br/>
+
Fig 1. Construction map for experimental main construct<br>
 +
•  K576005 contains the first component of GFP (GFPL)<br>
 +
•  K576003 contains the first part of the intron sequence (INL)<br>
 +
•  J61046 contains the lox site<br>
 +
•  K576006 contains the second component of GFP (GFPR)<br>
 +
•  K576004 contains the second part of the intron sequence (INR)<br>
 +
•  K576007 contains GFPL and INL<br>
 +
•  K576009 contains GFPL, INL and lox1<br>
 +
•  K576011 contains the promoter (P), ribosomal binding sit (RBS), GFPL, INL, lox site, INR, GFPR and transcriptional terminator (TT). This is the final construct (experimental design)<br><br>
-
<h5>Wednesday, June 1, 20111</h5>
+
<img src="https://static.igem.org/mediawiki/2012/f/f0/UW2012-N02.png" alt="N01"></img><br>
-
<li>Created a frozen stock of I20260</li>
+
Fig 2. Construction map for positive and negative controls<br>
-
<li>Inoculated BBa_I0500 and BBa_E0240 from frozen stock already made from last year</li><br/>
+
•  K576005 contains the first component of GFP (GFPL)<br>
 +
•  K576006 contains the second component of GFP (GFPR)<br>
 +
•  K576013 contains the promoter (P), ribosomal binding site (RBS), GFP and transcriptional terminator (TT). This is the positive control.<br>
 +
•  K576005 contains the first component of GFP (GFPL))<br>
 +
•  J61046 contains the lox site<br>
 +
•  K576006 contains the second component of GFP (GFPR)<br>
 +
•  K576012 contains the promoter (P), ribosomal binding site (RBS), GFPL, lox site, GFPR and transcriptional terminator (TT). This is the negative control.<br><br>
-
<h5>Thursday, June 2, 20111</h5>
+
-
<li>Miniprepped and nanodropped I0500, and E0240.</li>
+
</div></div>
-
<li>Inoculated I20260 from the frozen stock created the previous day</li><br/>
+
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 +
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-
<h5>Friday, June 3, 20111</h5>
+
<b>Lab book: Main construct</b>
-
<li>Digestion Reaction. Digested I0500 with EcoRI and SpeI. Digested E0240 (first sample) with EcoRI and PstI and digested E0240 (second sample) with EcoRI and XbaI.</li>
+
<br><br>
-
<li>Miniprepped and nanodropped I20260</li><br/>
+
Week of June 17th-24th
 +
June 17th: First general meeting with lab volunteers, addressing general lab etiquettes and safety procedures. Explained the general lab working outline and project design. Exchanged contact information and constructed a lab schedule for volunteers and lab leaders.  
 +
<br><br>
-
<h5>Monday, June 6, 20111</h5>
+
June 18th: Lab leader (Emily) inoculated parts 118-PSB1C3 backbone(Cmr), 119-InL(Ampr), 120-InR(Ampr), 121-GFPR(Ampr),122-GFPL(Ampr) and 123-Lox (Ampr). All parts except 118 are in puc57, which is ampicillin resistant. Cultures inoculated in LB broth, incubated at 37° C overnight.
-
<li>Inoculated I20260, I0500 and E0240</li><br/>
+
<br><br>
-
<h5>Tuesday, June 7, 20111</h5>
+
June 19th: Volunteers invited into the lab with a brief opening and orientation. Two volunteers trained and conducted miniprep of overnight cultures. Samples are quantified using Nanodrop Spectrophotometer
-
<li>Miniprepped and nandodropped I20260, and I0500 and E0240 for extra sample</li>
+
<br><br>
-
<li>Inoculated E0240</li><br/>
+
-
<h5>Wednesday June 8, 20111</h5>
+
June 24th: Digested samples (118-122) with Fermentas Fast-digest restriction enzymes EcoR1 and Pst1 to subclone parts 119-122 into PSB1C3 (118). Digestion was incubated at 37° C for an hour (may be too long, but no star activities detected).
-
<li>Digestion reaction. Digested I0500 with EcoRI and SpeI. Digested E0240 (first sample) with EcoRI and PstI and digested E0240 (second sample) with EcoRI and XbaI. Digested I20260 with EcoRI and PstI.</li>
+
<br><br>
-
<li>Miniprepped and nanodropped E0240 for back up</li><br/>
+
-
<h5>Thursday June 9, 20111</h5>
+
A diagnostic gel then follows by loading the cut samples along with the uncut minipreps for comparison.  
-
<li>Gel extraction of E0240 (EcoRI+XbaI), I0500 (EcoRI+SpeI), E0240 (EcoRI+PstI) and I20260 (EcoRI+PstI). Two samples of each parts were gel extracted.</li>
+
<br><br>
-
<li>Nanodropped the samples from Gel extraction. Concentration of I0500 and I20260 were too low.</li>
+
-
<li>Inoculated I0500 and I20260</li><br/>
+
-
<h5>Friday June 10, 20111</h5>
+
129bp band observed for InL (119), 172bp observed for InR (120). 545bp and 399bps are observed for GFPL (122) and GFPR (121) respectively. Differences observed between the undigested and digested samples.  
-
<li>Miniprepped I0500 and I20260 and ran digestion reaction. Same enzymes were used as the ones listed above.</li><br/>
+
<br><br>
-
<h5>Tuesday, June 14, 20111</h5>
+
June 27th:  Run the sample gel as last time but with no control. Gel extracted samples using Biobasic and fermentas binding buffers.  
-
<li>Gel extraction of I0500, I20260 and the back-up samples of E0240.</li><br/>
+
<br><br>
-
<h5>Thursday, June 16, 20111</h5>
+
June 28th: Nanodrop results show very low concentration (less than 1uL) for InL and 2. Gel extraction need to be repeated
-
<li>Ligation reaction: Ligated I0500 on to E0240. Ligated I20260 on to pSB1A2 (from E0240)</li><br/>
+
<br><br>
-
<h5>Monday, June 20, 20111</h5>
+
June 29th: Gel extraction repeated and concentration greatly increased. Enough for ligation
-
<li>Transformation of ligated parts (I0500+E0240, and I20260 on pSB1A2). Plated the transformant and incubated overnight</li><br/>
+
<br><br>
-
<h5>Tuesday, June 21, 20111</h5>
+
July 4th: Ligation using 1:3 vector to insert ratio in moles. InL, InR, GFPL and GFPR are ligated into PSB1C3 with vector+ ligase control and vector only control. The ligation tubes are incubated overnight at room temperature.  
-
<li>Created plates containing 1% arabinose.</li>
+
<br><br>
-
<li>Replicated plates containing I20260 colonies on to a different plate with Ampicilin antibiotic. Incubated overnight.</li><br/>
+
-
<h5>Wednesday, June 22, 2011</h5>
+
July 5th: Transformation into DH5a cells using heat shock technique. Cells plated on LB Cmr plates and incubated overnight at 37° C. Added transformation control (vector uncut into DH5a) and negative control (DH5a cells only). 1/10 and 9/10 dilutions for each ligation
-
<li>Replicated I0500+E0240 to plates containing 1% arabinose. Incubated overnight</li>
+
<br><br>
-
<li>Inoculated a single colony from I20260 plate</li><br/>
+
-
<h5>Thursday, June 23, 2011</h5>
+
July 6th: Took out transformation plates. Pink colonies (hundreds) only observed on vector+ligase, vector only controls and transformation controls. Negative control plates are clean. Plates with insert and plasmid have hundreds of white colonies and some pink colonies present. Two morphologies present; large and small
-
<li>Created frozen stock of I20260</li>
+
<br><br>
-
<li>Inoculated a single colony from I0500+E0240 plate</li><br/>
+
-
<h5>Friday, June 24, 2011</h5>
+
July 9th: Streak purified 24 white colonies selectively (6 of each 3 large, 3 small)
-
<li>Created frozen stock of I0500+E0240</li><br/>
+
<br><br>
-
<h5>Monday, July 4, 2011</h5>
+
July 10: Inoculate and patched colonies into LB CmR tubes/plates
-
<li>Inoculated the I20260, and I0500+E0240</li>
+
<br><br>
-
<li>Created 1M Arabinose solution</li><br/>
+
-
<h5>Tuesday, July 5, 2011</h5>
+
July 11: Picked 8 final colonies to screen (1 big and 1 small each set) Miniprep these samples
-
<li>Made serial dilutions of arabinose solution (from 1M to 1uM).</li>
+
<br><br>
-
<li>Diluted the cultures to 0.50 Absorbance at OD600.</li>
+
-
<li>Set up experiment for quantification experiment. Refer to the diagram below for how the experiment was setup.</li><br/>
+
-
<img src="https://static.igem.org/mediawiki/2011/f/f8/Waterloo_Quantification.png" width= "500" height="250"></img>
+
-
<br/><br/>
+
-
<h5>Monday, July 11, 2011</h5>
+
July 12: Digested samples to check for insert (with EcoR1 and Pst1). Loaded a diagnostic gel afterwards
-
<li>Inoculated the I20260, and I0500+E0240</li>
+
<br><br>
-
<li>Created 1M Arabinose solution</li><br/>
+
-
<h5>Tuesday, July 12, 2011</h5>
+
Result: Proper sized inserts were observed for all samples. All parts were subcloned successfully into PSB1C3
-
<li>Another quantification experiment run. Machine failure.</li><br/>
+
<br><br>
-
<h5>Wednesday, July 13, 2011</h5>
+
July 13: Inoculated for more miniprep
-
<li>Inoculated the I20260, and I0500+E0240</li>
+
<br><br>
-
<li>Created 1M Arabinose solution</li><br/>
+
-
<h5>Thursday, July 14, 2011</h5>
+
July 14: Miniprep samples again and saved frozen stock of samples
-
<li>Last quantification experiment run.</li><br/><br/><br/>
+
<br><br>
 +
July 18th: Digestion of InL and GFPL with BgIII and EarI (sites overlap, put EarI in first and add BgIII after 30 minutes). Insert (115bp) observed for digestion with InL
 +
<br><br>
 +
July 19th: Gel extracted InL and GFPL
 +
<br><br>
-

<b>The following entries pertain to the Ribozyme Project</b><br/><br/>
+
July 21st: Ligated InL with GFPL with 1:3 insert: vector ratio in moles. Incubated at room temperature overnight
-
<h5>Wednesday July 6, 2011</h5>
+
<br><br>
-
<li>Received 3 of 4 sequences the previous week (IN1, IN2 and GFP2).</li>
+
-
<li>A quick spin down UWAT014-3/UWAT014-2 using centrifuge.</li>
+
-
<li>Resuspended DNA in 40ul of MQ water (Concentration: 2ug/40ul=1ug/20ul)</li>
+
-
<li>Transformed into DH5-alpha (sequences in PUC57).</li>
+
-
<li>grown overnight on ampicillin plates.</li>
+
-
<li>Resuspension of PSB1C3 in the Spring 2011 distribution kit (Plate 1 well 3A). Contains BBa_J04450.</li>
+
-
<li>Resuspension of PSB1C3 in 10ul of MQ water (aspirated), wait approximately 5 minutes.</li>
+
-
<li>1ul of resuspension was transformed into DH5-alpha. Grown overnight on plate.</li><br/>
+
-
<h5>Thursday July 7, 2011</h5>
+
July 23rd: Transformed ligation product with transformation and negative control on LB Cm plates
-
<li>N1 (amp), IN2 (amp), GFP2 (amp) and PSB1C3 (cm) broth cultures innoculated (3 each)..</li><br/>
+
<br><br>
-
<h5>Friday July 8, 2011</h5>
+
-
<li>Frozen stock of IN1, IN2 and GFP2 in PUC57 and PSB1C3 backbone made..</li>
+
-
<li>Miniprep for IN1, IN2, GFP2 and PUC57 completed:.</li><br/>
+
-
<table border="1">
+
July 24th: All cells had a lawn of cells.  
-
<tr><td><b>Sequences</b></td><td><b>In1</b></td><td><b>In2</b></td><td><b>GFP 2</b></td><td><b>pSB1C3</b></td></tr>
+
<br><br>
-
<tr><td><b>260/280</b></td><td>1.85</td><td>1.80</td><td>1.88</td><td>1.86</td></tr>
+
-
<tr><td><b>ng/ul</b></td><td>229.8</td><td>236.1</td><td>198.6</td><td>166.2</td></tr></table><br/>
+
-
<li>GFP1 Sequence(588nt)in PUC57 received from Bio Basic Canada INC..</li><br/>
+
-
<h5>Tuesday July 12, 2011</h5>
+
-
<li>Liquid cultures of GFP1 (x2), IN1, IN2, GFP2 and PSB1C3 were innoculated with the appropriate antibiotic in the broth..</li><br/>
+
-
<h5>Wednesday July 13, 2011</h5>
+
-
<li>GFP1, IN1, IN2, GFP2 and PSB1C3 were minipreped to isolate plasmid DNA..</li>
+
-
<li>Frozen stock of GFP1 made..</li><br/>
+
-
<h5>Thursday July 14, 2011</h5>
+
-
<li>GFP1, IN1, IN2, GFP2, PSB1C3 digested with EcoRI and PstI. GFP2 also digested with ndeI..</li>
+
-
<li>Innoculation of liquid culture (GFP1, IN1, IN2, GFP2, PSB1C3)..</li><br/>
+
-
<h5>Friday July 15, 2011</h5>
+
-
<li>Gel extraction of GFP1, IN1, IN2, GFP2 and PSB1C3. However, the results were not as anticipated..</li>
+
-
<li>Miniprep of cultures innoculated yesterday.</li><br/>
+
-
<h5>Monday July 18, 2011</h5>
+
-
<li>Cultures were miniprepped, however, GFP1 did not have a sufficient concentration to undergo digestion..</li>
+
-
<li>Proceeded with digestion for GFP2 (ndeI), IN1, IN2 and PSBIC3 with EcoRI and PstI..</li>
+
-
<li>Innoculation of GFP1 (x4).</li><br/>
+
-
<h5>Tuesday July 19, 2011</h5>
+
-
<li>Miniprep and digestion of GFP1..</li>
+
-
<li>Gel extraction of each digestion (PSB1C3(x2), GFP1, IN1, IN2, GFP2)..</li><br/>
+
-
<h5>Wednesday July 20, 2011</h5>
+
-
<li>Lox resuspended and digested..</li>
+
-
<li>Ligation of GFP2, IN1, IN2 and Lox into PSB1C3..</li><br/>
+
-
<h5>Thursday July 21, 2011</h5>
+
-
<li>Transformation of GFP2, IN1, IN2 and Lox. Each was plated on cm containing media and grown overnight..</li><br/>
+
-
<h5>Friday July 22, 2011</h5>
+
-
<li>All negative plates did not produce colonies..</li>
+
-
<li>Growth was good on all positive plates except for IN1, which only produced two main colonies..</li><br/>
+
-
<h5>Monday July 25, 2011</h5>
+
-
<li>Gel extraction IN1, IN2 and GFP1, however, GFP1 failed..</li>
+
-
<li>Ligation of IN1 and IN2 into PSB1C3..</li><br/>
+
-
<h5>Tuesday July 26, 2011.</h5>
+
-
<li>Innoculation of GFP2 into cm containing LB broth tube and lox into amp containing LB broth tube..</li>
+
-
<li>Gel extraction did not work (likely a problem with digestion or transformation).</li>
+
-
<li>Innoculation x5 of GFP1..</li><br/>
+
-
<h5>Wednesday July 27, 2011</h5>
+
-
<li>Frozen stock of GFP2 and lox made.</li>
+
-
<li>Miniprep of GFP1 x4 replicates.</li>
+
-
<li>Gel extraction of IN1 and IN2 resulted in improved concentrations.</li>
+
-
<li>Transformation.</li><br/>
+
-
<h5>July 30, 2011</h5>
+
-
<li>Miniprepped GFP 1 and GFP 2.</li><br/>
+
-
<h5>August 2, 2011</h5>
+
-
<li>Miniprepped Intron 1 and intron 2..</li><br/>
+
-
<h5>August 3, 2011</h5>
+
-
<li>Digestion of GFP 1 and Intron 2 with SacI and EarI.</li>
+
-
<li>Digestion of GFP2 and Intron 1 with SacI and SapI.</li><br/>
+
-
<h5>August 4, 2011</h5>
+
-
<li>Nothing.</li>
+
-
<li>Figuring out unexpected SacI cute site in the middle of pSB1C3 vector.</li><br/>
+
-
<h5>August 5, 2011</h5>
+
-
<li>Nothing.</li><br/>
+
-
<h5>August 6, 2011</h5>
+
-
<li>Digestion of GFP 1 with Bgl II and Ear I.</li>
+
-
<li>Digestion of Intron 1 with Bgl II and Ear I.</li>
+
-
<li>Digestion of GFP 1 with Ear I and Pst I.</li>
+
-
<li>Digestion of GFP 2 with EarI and Pst I..</li>
+
-
<li>Gel extraction of all the samples listed above.</li><br/>
+
-
<h5>August 7, 2011</h5>
+
-
<li>Ligation of GFP 1 with Intrton 1 and GFP 1 with GFP 2.</li>
+
-
<li>Transformation of the two ligation mixtures listed above.</li><br/>
+
-
<h5>August 8, 2011</h5>
+
-
<li>Minipreping Int2, lox and GFP2.</li>
+
-
<li>Digestion.</li>
+
-
<li>Gel extraction.</li><br/>
+
-
<h5>August 9, 2011</h5>
+
-
<li>Miniprepping BBa_K576007.</li>
+
-
<li>Digestion.</li>
+
-
<li>gel extraction.</li><br/>
+
-
<h5>August 10, 2011</h5>
+
-
<li>Ligation and transformation of BBa_K576007 and J61046 to create K576009.</li><br/>
+
-
<h5>August 11, 2011</h5>
+
-
<li>Ligation and transformation.</li><br/>
+
-
<h5>August 12, 2011</h5>
+
-
<li>Streak plating GFP1-GFP2 (BBa_K576013).</li>
+
-
<li>Backbone for RFC arrived and was streaked on cm plate.</li><br/>
+
-
<h5>August 15th, 2011</h5>
+
-
<li>Inoculation of a single colony of K371053 into an LB broth</li><br/>
+
-
<h5>August 16th, 2011</h5>
+
July 26th: Miniprep and digested using the same protocol as above
-
<li>Made frozen stock of K371053</li>
+
<br><br>
-
<li>Inoculated K371053 (X3), K576004, and K576006.</li><br/>
+
-
<h5>August 17th, 2011</h5>
+
July 27thh: Miniprep PSB1C3
-
<li>Miniprepped K371053(vector), K576004 (insert),  and K5756006 (insert).</li>
+
<br><br>
-
<li>Standard assembly of the two parts and a vector listed above. Ran gel extraction experiment after.</li><br/>
+
-
<h5>August 18th, 2011</h5>
+
July 28th: Miniprep lox and digested lox using Xbal and Pst1. Digested lox loaded onto 2% gel.
-
<li>Ligation and transformation reaction for transferring K576004 and K576006 from pSB1C3 to K371053.</li><br/>
+
<br><br>
-
<h5>August 19th, 2011</h5>
+
August 2nd: Gel extracted InL and GFPL again for ligation and conducted ligation again
-
<li>Inoculation of single colonies from the transformed samples from the previous days.</li>
+
<br><br>
-
<li>Inoculated K576009</li><br/>
+
-
<h5>August 22nd, 2011</h5>
+
August 9th: Transformation into DH5a
-
<li>Created frozen stock and inoculated K576004 and K576006 on K371053</li>
+
<br><br>
-
<li>Miniprepped, digested (SpeI and PstI) and gel extracted K576009.</li><br/>
+
-
<h5>August 23rd, 2011</h5>
+
August 13: Repeat miniprep of InL and GFPL
-
<li>Miniprepped, digested and gel extracted K576004 (SpeI and PstI) and K576006 (XbaI and PstI) on K371053. </li><br/>
+
<br><br>
-
<h5>August 24th, 2011</h5>
+
August 14th: Digested InL and GFPL with EarI and BgIII again
-
<li>Ligation and transformation of samples prepared on the 23rd. </li><br/>
+
<br><br>
-
<h5>August 25th, 2011</h5>
+
Exam break
-
<li>Single colony for K576008 was picked and inoculated</li><br/>
+
<br><br>
-
<h5>August 26th, 2011</h5>
+
August 28th: Final ligation of InL and GFPL
-
<li>Created frozen stock of K576008</li><br/>
+
<br><br>
-
<h5>August 28th, 2011</h5>
+
August 29th: Transformation. All colonies are white, but more colonies present for the inesrt than vector + ligase only. 
-
<li>Inoculated K576008 and K576009 in LB broth containing chloramphenicol</li><br/>
+
<br><br>
-
<h5>August 29th,2011</h5>
+
September 17:  Picked colonies for inoculation
-
<li>miniprepped and digested K576008 and K576009.</li><br/>
+
<br><br>
-
<h5>August 30th, 2011</h5>
+
September 18: Miniprep of samples
-
<li>Gel extraction of K576008 (insert) and K576009 (vector). </li><br/>
+
<br><br>
-
<h5>August 31st, 2011</h5>
+
September 19: Digestion of samples and loading of gel. No inserts present.
-
<li>Ligation and transformation of final construction (BBa_K576011)</li><br/>
+
<br><br>
-
<h5>September 1st-2nd, 2011</h5>
+
<b>Lab book: Controls</b><br><br>
-
<li>No lab work was done.</li><br/>
+
-
<h5>September 4th, 2011</h5>
+
June 21st 2012: Enzyme digestion and gel extraction of pSB1C3, BBa_K576005 in pUC57, BBa_K576006 in pUC57 for subcloning. There were unexpected bands on the gel we will have to redo experiment. <br><br>
-
<li>inoculated K576011 and K576008 in LB broth containing Chloromphenicol antibiotic</li><br/>
+
-
<h5>September 5th, 2011</h5>
+
June 29th 2012: Enzyme digestion and gel extraction of pSB1C3, BBa_K576005 and BBa_K576006 for subcloning. <br><br>
-
<li>Miniprepped, digested (EcoRI and PstI) and gel electrophoresed part K576008 to move the part into pSB1C3 for submission. Questionable band placement; Did not go further with the experiment.</li>
+
-
<li>Miniprepped all the intermediate steps, controls and final construction for submission to partsregistry.</li><br/>
+
-
<h5>September 6th, 2011</h5>
+
July 7th 2012: Ligation of BBa_K576005 into pSB1C3 and ligation of BBa_K576006 into pSB1C3 for subcloning. <br><br>
-
<li>Miniprepped, digested and gel extracted part K576008. Confirmed band placement via aPe program. Failed at the final step of the gel extraction process; Wash Solution without ethanol (miniprep solution provided from Biobasic) was added </li><br/>
+
-
<h5>September 7th, 2011</h5>
+
July 10th 2012: Transformation of BBa_K576005 in pSB1C3 and BBa_K576006 in pSB1C3 into DH5-alpha for subcloning.  <br><br>
-
<li>Miniprepped, digested and gel extracted part K576008. </li><br/>
+
 
 +
July 11th 2012: Checked the plates and they were all lawns. We will use Emilys subcloned parts for now. <br><br>
 +
 
 +
July 17th 2012: Inoculation of pSB1C3 containing strain and BBa_J61046 in pUC57 containing strain. <br><br>
 +
 
 +
July 18th 2012:  Our frozen stock may be contaminated so we discarded the inoculation tubes from July 17th and we will use Emilys miniprepped samples for now. Restriction digest and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006.  <br><br>
 +
 
 +
July 21st 2012: Ligation to make BBa_K576013 construct. <br><br>
 +
 
 +
July 22nd 2012: Transformation of BBa_K576013 construct into DH5-alpha. <br><br>
 +
 
 +
July 23rd 2012: All the plates were lawns. Shaking the broth we used showed it was contaminated. Discard the plates and repeat the experiment.  <br><br>
 +
 
 +
July 28th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. The bands on the gel looked as expected but the nanodrop values after the gel extraction were indicated we did not have DNA. We will repeat the digest and gel extraction using isopropanol to increase yield.    <br><br>
 +
 
 +
August 18th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Ligation to make BBa_K576013 construct. <br><br>
 +
 
 +
August 19th 2012: Transformation of BBa_K576013 construct into DH5-alpha. Transformation of left construct intermediate for the main construct. <br><br>
 +
 
 +
August 20th 2012: All the plates were lawns. This time we are confident the broth was not contaminated. Our problem could be with the antibiotic. Next time we will plate a 1/10 dilution of the cells. <br><br>
 +
 
 +
August 21st 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006.  The bands on the gel looked as expected but the nanodrop values after the gel extraction were not good enough to move onto ligation. <br><br>
 +
August 26th 2012:  Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Restriction digestion of pSB1C5. pSB1C5 showed an unexpected band in the gel so we did not proceed to gel extraction.  <br><br>
 +
 
 +
August 28th 2012: Ligation to make BBa_K576013 construct. <br><br>
 +
 
 +
August 29th 2012: Transformation of BBa_K576013 construct into DH5-alpha. Transformation of left construct intermediate for main construct into DH5-alpha. <br><br>
 +
 
 +
August 30th 2012: All the plates were clean except the transformation of the miniprepped sample for a positive control had colonies. Our problem is therefore not with the transformation. Our problem may be with our ligation. We will repeat the experiment with an additional control for the ligation. <br><br>
 +
 
 +
September 6th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Linearize and gel extract pSB1C3 for additional ligation control. Ligation to make BBa_K576013 construct and ligation of linearized pSB1C3. <br><br>
 +
 
 +
September 7th 2012: Transformation of BBa_K576013 construct into DH5-alpha. <br><br>
 +
 
 +
September 8th 2012: The transformed miniprep and transformed ligated pSB1C3 had colonies. The rest of the plates were clean. Therefore the transformation and ligation was successful however the construct still didn't work out. We will repeat the experiment after miniprepping more BBa_K576006 in pSB1C3. Inoculate BBa_K576005 in pSB1C3.  <br><br>
 +
 
 +
September 9th 2012: Miniprep BBa_K576006 in pSB1C3. <br><br>
 +
 
 +
September 11th 2012: Streak purify DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576005 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 from frozen stock.  <br><br>
 +
 
 +
September 12th 2012: Inoculate DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576005 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 in Terrific broth. <br><br>
 +
 
 +
September 13th 2012: Miniprep BBa_K576005 in pSB1C3, BBa_K576006 in pSB1C3 and BBa_J61046 in pUC57. <br><br>
 +
 
 +
September 15th 2012: Restriction digestion of parts BBa_K576005, BBa_K576006 and BBa_J61046 to get gel extracted fragments for both the positive and negative control constructs. Diagnostic gel showed questionable bands so we need to repeat the digestion. <br><br>
 +
 
 +
September 16th 2012: Repeat the digestion from September 15th. This time the bands are as expected so the fragments were gel extracted. The nanodrop values of gel extracted BBa_K576005 in pSB1C3 for the negative control construct indicated we did not have DNA so need to redo that sample. Other samples were stored. <br><br>
 +
 
 +
September 18th 2012: Restriction digestion of BBa_K576005 in pSB1C3 for negative control construct but the gel showed an unexpected band so did not gel extract. Ligation to make BBa_K576013 construct. Inoculation of DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576006 in pSB1C3,  DH5-alpha carrying BBa_K576003 in pSB1C3, DH5-alpha carrying BBa_K576004 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 in Terrific broth. <br><br>
 +
 
 +
September 19th 2012: Miniprep of BBa_K576005 in pSB1C3, BBa_K576006 in pSB1C3, BBa_K576003 in pSB1C3, BBa_K576004 in pSB1C3 and BBa_J61046 in pUC57. Transformation of BBa_K576013 construct into DH5-alpha. <br><br>
 +
 
 +
September 20th 2012: All plates were clean except the transformed miniprep. Stop lab work on BBa_K576013 construct due to time constraints. <br><br>
 +
 
 +
September 23rd 2012: Restriction digestion of minipreps from 1-12 colonies to screen left construct transformation plate. Load diagnostic amounts of digested samples to image BBa_K576005, BBa_K576006, BBa_K576003, BBa_K576004 and BBa_J61046 bands in a gel to demonstrate successful subcloning. The BBa_K576003 and BBa_K576004 bands were not visible in the gel. <br><br>
 +
 
 +
September 24th 2012: Load diagnostic amounts of digested miniprepped 1-10 colonies to screen left construct plate.  <br><br>
-
<h5>Septermber 8th, 2011</h5>
 
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<li>Ligation and transformation reaction for K576008. Moved K576008 part from the BBa_K371053 to pSB1C3 for submission purposes.</li><br/>
 
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<h5>Septermber 9th – 15th, 2011</h5>
 
-
<li>no lab work was done</li><br/>
 
-
<h5>September 16th, 2011</h5>
 
-
<li>Final diagnostic digestion reaction. Digested every intermediate, control and final constructs. BBa_K576003, K576004, K576005, and K576006 were the only parts able to be confirmed. All the other intermediates and constructs have questionable band location. Lab work stopped due to time constraint. </li><br/><br/>
 
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In Vivo Protein Fusion Assembly Using Self Excising Ribozymes

ABSTRACT

Waterloo's 2012 iGEM project is a continuation of the 2011 project, In Vivo Protein Fusion Assembly Using Self Excising Ribozymes. This year our hope is to complete the project with the aim of potentially designing future projects which incorporate this system.

Self-excising ribozymes are RNA sequences with catalytic properties which allow them to remove themselves and the regions which they flank from an RNA sequence. These are introns; however, with ribozyme self-excision the introns are removed without the aid of protein enzymes. In our project we use self-excising ribozymes to remove an extraneous sequence, an intron, which interrupts the coding sequence of GFP. Upon successful removal of the intron, the two halves of GFP should be ligated together and be able to be translated into a fully functional GFP. By showing that functional fusion proteins can be assembled in-vivo using this system we open up possibilities such as the addition of recombination sites to allow gene shuffling, and regulatory sequences which function at the DNA level but that are removed at the RNA level to create functional proteins.




WE WOULD LIKE TO THANK OUR GENEROUS SPONSORS.


Faculty of Science
The Intron
Project
Outreach
Human
Practices
The Intron Project

      with Mathematical Modelling




1.0 Abstract

The Group 1 introns are a widely spread group of ribozymes. Many of them exhibit self-excising activity. These could be used to generate in vivo recombination libraries by incorporating them with a Cre-lox system, which can facilitate chromosomal gene transposition. Below we outline a proof-of-concept experiment to display the feasibility of this experiment.


2.0 Introduction

2.1 Cre-lox system

The Cre-lox system is an in-vivo recombination system developed from P1 bacteriophage that is perhaps most familiar to students as a method of gene self-excision (1). The Cre enzyme is a site specific recombinase. It recognises loxP sites in DNA (34bp), and will either excise, invert or translocate DNA between them (Fig 1). The Cre enzyme may be designed for expression under specific conditions (eg. IPTG induction), allowing some control over in vivo recombination. The caveat to using any recombination system to generate fusion proteins in vivo, is that translation of the leftover recombination site sequence or 'scar' can shift codon readings, and/or disrupt protein folding (Fig 2) We propose, therefore, to flank a recombination site, such as the loxP sequence, with a self-excising ribozyme sequence, to create 'scarless' fusion proteins (Fig 2).

Fig 1

Fig 1. Schematic of Cre-lox recombination (from (2))



Fig 2
Fig 2. Schematic of recombination products a) with a remaining 'scar' and b) with the scar removed from between two fusion protein segments, PL and PR. Failure to remove the lox scar may result in disrupted protein folding or mistranslation of the second half of the protein, PR




2.2 Group I Introns

Self-excising ribozymes are an ancient class of introns that can remove themselves from RNA constructs, including mRNA and tRNA (3). The Group I introns are a subset of these self-excising ribozymes, and are widely distributed through simple eukaryotes, fungi, mitochondria, chloroplasts, bacteria and bacteriophage (3). Group I introns are not yet known to provide a specific biological function, save to excise themselves from important RNA sequences, and thus prevent host death. One hypothesis is that they are "selfish" remnants of an RNA world (4). Some Group I introns, for example, code for DNA endonucleases that help them migrate to new sites (3).

The Group I excision mechanism relies on a reactive 'core' rich in RNA secondary structure that conducts two successive transesterifications (5). Some introns require proteins to help stabilise them, while others are completely independent (5). The first transesterification involves a nucleophilic attack on the 5' end of the intron, by the 3' OH of a guanosine nucleotide (5) (Fig 3). Structural rearrangements bring the the 3' end of the exon into proximity with the 3' end of the intron, allowing the second transesterification to occur (5) (Fig 3). The intron is usually then degraded by the host, while the resulting mRNA is translated.

Fig 3
Fig 3. Mechanism of intron self-excision (adapted from (5)) – The construct used above is our experimental construct, further discussed below. (a) The first transesterification occurs when a GMP attacks the 5' end of the intron (InL-lox-InR). (b) The second transesterification occurs at the 3' end of the intron, led by the 3' end of the exon. This results resulting in (a) the fusion mRNA for transcription and (d) the intron sequence, to be degraded.




2.3 Staphylococcus phage twort ORF142

The Staphylococcus aureus bacteriophage Twort is notable for having three introns within a single gene (6). This ORF142 is a putative structural protein, though its role has not yet been confirmed. Sequence analysis reveals three highly similar introns, I1, I2 and I3, closely interspersed in the gene. Experiments show that they are self-excising, and can produce variable splice products, depending on the intron(s) removed (6). This is likely due to their terminal sequence similarity (6). We have chosen to work with a modified version of these introns due to their independent, self-excision capability, and the low likelihood of disrupting important secondary structure on incorporation of additional RNA.

2.4 Application

Gene shuffling has been used to create fusion protein libraries of compounds that are more effective than the parent proteins. Examples include interferon, antibodies and Cry proteins, a family of biological insecticides (7). Many of these recombination systems, however, rely on in vitro recombination, cloning, then expression and screening of the protein products. An in vivo recombination system allows the recombination and expression to happen in the same step.

3.0 Lab and Design

Our project, which continues from last year, is a proof-of-concept for the use of Group I introns in providing scarless removal of an internal sequence. The marker gene we are using is GFP. Fig 3 (above) and Fig 7 (below) describe the desired, final construct. Essentially, successful self-excision of the intron should result in a normal, functional GFP protein. The final construct will be assembled by joining five "pieces": the two halves of the GFP protein, the two halves of the intron, and a lox recombination site.

3.1 Mathematical model

In order to characterize the relative efficiency of the intron system, a model was developed based on the idea proposed by Kelly et al (8) of comparing steady state fluorescence to a common standard. Our model develops a ratio of protein expression in a system with the intron (Pintron) (Fig 4), to expression in a system without the intron (PGFP) (Fig 5). It assumes that all reaction rates are linear, and that they remain constant between cell populations. If the rate of mRNA degradation (δM) is known or estimated, one can approximate the rates of successful mRNA splicing (αS) and failed splicing or folding (αF). The resulting measure of efficiency of splicing, then, is αS / (αS + αF)

Fig 4
Fig 4. Model predicting amount of protein formed from a construct containing an intron

Fig 5
Fig 5. Model predicting amount of protein formed from an intron-less construct

Fig 6
Fig 6. The ratio of the rate of protein synthesis with and without an intron


3.2 Procedure outline

We have designed the construct to be built using the meta format of RFC53 in pSB1C3 and pSB1C5. RFC53 is useful for creating scarless recombination between DNA sites using the REN EarI. This cleaves a set distance downstream of their recognition site.

First, the pieces must be subcloned from pUC57 into pSB1C3 or pSB1C5, which contain the meta-suffix format of RFC53. The planned construct maps are shown in Fig 7.

Fig 7
Fig 7. Planned constructs (a) main experimental construct (K576011), (b) positive control (K576013), (c) negative control (K576012) in pSB1C3


Last year, a diagnostic of the final ligation products revealed unexpected banding. This year, we have re-done the subcloning into pSB1C3, and have proceeded to attempt the ligation of the final construct with more caution. Previously, diagnostic gels were run only insofar as gel extraction had to be done. As such there was no confirmation of the ligation pieces before joining them. This was in part due to the size of the fragments, as several of them are quite small, making it difficult sometimes to visualize them on a gel.

3.3 Lab work, results and conclusions

This year, in addition to conduct the expected laboratory research on the intron project, we invited 20 undergraduate students from the University of Waterloo to join us, in order to teach them some basic molecular biology techniques. Volunteers learned simple molecular biology techniques such as restriction enzyme mapping, miniprep plasmid samples, vector cloning, heat-shock transformation and agarose gel extraction.

The original parts received by our team include left intron (aka Intron 1), right intron (Intron 2), left GFP (GFP 1), right GFP (GFP 2) and a lox region of 34 bp in length. All parts were cloned into pUC57 vector backbone, which is ampicillin resistant. In order to conduct the RFC 53 protocol in our cloning procedure, we need to first sub-clone the R-Intron, L-Intron, L-GFP and R-GFP into pSB1C3 backbone (Chloramphenicol resistant or CmR).

To complete the sub-cloning process, we followed a flow-chart as shown in Figure 8(a)

Fig 8a
Fig 8a

We used REN EcoR1 and Pst1 to double digest the original parts and then cloned into pSB1C3 vector. We transformed the cloned vector + insert samples into competent DH5α strain of Escherichia coli and then plated the colonies onto plates with Chloramphenicol.

Controls are important for plasmid cloning; in addition to our transformation plates, we included vector+ligase control, vector-only control, transformation control and negative (no plasmid) control to check for possibility of contamination and improper techniques. As expected, the transformation control, vector+ligase and vector-only controls had only pink colonies growing on them. The negative control plates were clean.

Our transformation plates showed a mix of pink and white colonies; the pink colonies represent the presence of Red fluorescent Protein in pSB1C3. As the multiple cloning site for pSB1C3 lies inside a red fluorescent protein (RFP) gene, successful transformants are white due to disruption of the RFP. We screened 8 of these colonies (2 colonies for each type of insert) and checked the size of the inserts by running an agarose gel, as shown in Figure 8 (b). In addition, we also loaded the digested pUC57 samples to ensure the inserts look identical. The ladder we used in this agarose gel is Fermentas GeneRuler 1kb DNA ladder. We have saved 2 copies of each of 8 clones in our lab freezer and we have also sent one of each type of clone (a total of 4 clones) to the IGEM registry.

Fig 8b
Fig 8b: Sub-cloning results with Fermentas GeneRuler 1kb DNA ladder


After successfully cloning the inserts into pSB1C3, we proceed with the RFC 53 protocol to make the main construct. Unfortunately, we were unable to find a clone with the right-sized insert for the L-intron and L-GFP ligation due to time constraints. As a result, we were unable to finish the making of the main construct.

The positive control construct is assembled using RFC 53. Fluorescence is expected to demonstrate that the one amino acid scar from RFC 53 does not disrupt GFP. The negative control construct is assembled using RFC 10. Fluorescence is not expected, in order to demonstrate that presence of the lox site disrupts GFP folding and activity.

Fig 8c
Fig 8(c): image of the gel used to extract fragments for the control constructs


Figure 8(c) is an example of a gel used to prepare gel extracted fragments for the two control constructs. We were unable to get any successfully transformed colonies for either construct.

Two criticisms in last year's experimental design have been highlighted this year, which may explain some of the difficulties in creating a working construct. First, many of the fragments being used are relatively small, which makes it difficult to isolate them for ligation. A suggested solution is to use PCR to amplify or introduce, for example, the 34bp lox site to the construct. Second, some of the REN being used for double digests are very close to each other on the plasmid (< 10bp). This may be impeding the RENs' ability to bind and cleave DNA. An alternative design to the one proposed here would be to create a gene with multiple intron sites to allow continuous mRNA recombination in vivo, rather than a one time recombination event at the DNA level (6).



3.4 Side project: Promoter expression quantification

In parallel with the lab work of constructing the intron system, progress was made at our lab towards implementing a framework for measuring relative fluorescence of cell cultures, using the protocol described by Kelly et al. (8). Such measurements are necessary in order to quantify the notion of intron efficiency developed in our model.

Work on this project is ongoing. The steps taken this summer were:
  • identification and acquisition of appropriate materials and equipment access
  • training of four undergraduate students, separate from the main lab project, in essential lab techniques
  • test-running the protocol and troubleshooting errors.

Once a quantification system is successfully implemented, our team will be also be equipped to begin making measurements of many other registry parts, according to the RPU standard proposed by Kelly et al. (8). Our vision is for RPU quantification of existing registry parts to be a standard component of UW iGEM's yearly activities, beginning with constitutive promoters and expanding into measurements of more complex devices.




References
(1) Nagy, A. (2000). Cre Recombinase: The Universal Reagent for Genome Tailoring. Genesis, 26(2), 99–109.
(2) The Jackson Laboratory. (n.d.). Introduction to Cre-lox technology. (http://cre.jax.org/introduction.html)
(3) Haugen, P., Simon, D. M., & Bhattacharya, D. (2005). The natural history of group I introns. Trends in Genetics, 21(2), 111–119.
(4) Lambowitz, A. M., Caprara, M. G., Zimmerly, S., & Perlman, P. S. (1999). The RNA World, Second Edition. New York: Cold Spring Harbor Laboratory Press, 451–486.
(5) Cech, T. R. (1990). Self-Splicing of Group I Introns. Annual Reviews in Biochemistry, 59, 543 – 568.
(6) Landthaler, M., & Shub, D. A. (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. Proceedings of the National Academy of Sciences of the United States of America, 96(12), 7005–7010.
(7) Glick, B. R., Pasternak, J. J., & Patten, C. L. (2010). Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition. Washington DC: ASM Press.
(8) Kelly, J. R., Rubin, A. J., Davis, J. H., Ajo-Franklin, C. M., Cumbers, J., Czar, M. J., de Mora, K., et al. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3:4.

UW iGEM OUTREACH PROJECTS 2011-12



Our Mission: Raise awareness on issues concerning synthetic biology to make informed, fact-based opinions.

Introduction

This year’s UW iGEM’s Outreach sub-team has learnt a lot from its experiences. We were met with a few hurdles with regards to planning and implementing the myriad of events we had hoped to have to engage our community. Though with every step there was much to be learnt and we have decided to discuss all the great events we did have planned and are in the process of planning. Along with that we thought it would be a great resource to create a guide that discussed various different types of outreach events you can hold and what hurdles you could potentially face (as we did) in terms of implementation.

Successes
Despite the barriers that we faced, we still had numerous successes. We were still able to develop Community Bricks for most of our programs so other Outreach members can implement them as well. The Community Bricks we were able to post include:
  1. 1. Grade 11 Synthetic Biology Workshop Program with Interactive Activities
  2. 2. DIY, At Home Gel Electrophoresis
  3. 3. Basic Lab Training Workshops
  4. 4. Communications Plan Template and Example for Your Large-Scale Outreach Programs
  5. 5. Tips and Tricks to Have a Successful Outreach Event

As well as…
Educational Video Podcast featuring the iGEM Outreach Lead Ekta Bibra on Synthetic Biology for Virtual Researcher On Call (organization providing educational resources to students and teachers through online web conferences or podcasts in Ontario, Canada). This material will be openly available for students and teachers to view and learn from across the province. This will be available for you to view at the regionals this year!
As well, we are working on a core social media campaign in which we amplify awareness with minimal costs, gain a larger audience base to ease overall promotion and easily apply Outreach’s over-arching goal of raising awareness on synthetic biology. Make sure you follow us and our progressions on:

Twitter: @Waterloo_iGEM

Facebook: Waterloo iGEM page

Outreach Activities


The divisions of our outreach events are regularly divided into seasonal timings, from Fall, Winter to Spring. This year these were the events that we had initially planned on implementing:

  1. BioTalks Open Panel Discussion and Lecture Series
    Community Brick (Communications Plan Plus Example)
    Purpose: Develop an exciting discussion forum between science undergraduates and industry specialists within biotechnology to encourage science entrepreneurship, innovation and industrial advice.
    How: Organize a large-scale open panel discussion involving brief 20 minute talks from key industry leaders followed by a question and answer period that can supplement what has been demonstrated within the short lectures.
    Type: Education/Awareness


  2. Engineering Science Quest (ESQ)
    View Community Brick
    Purpose: A tradition for the UW iGEM Outreach team to carry out, this workshop is held by ESQ to develop hands-on learning activities and workshops for students from Grade 3-12 to promote science/engineering education and outreach.
    How: Development of a do-it-yourself workshop for Grade 12 students that incorporates science into their everyday lives. Also created a Community Brick.
    Type: Education


  3. Hot Chocolate Event
    Purpose: To spread the word on campus of UWaterloo iGEM, gain presence amongst students of who we are and promote our social media accounts on Twitter and FaceBook.
    How: Selling hot chocolate for 50 cents along with a small paper tag of our FaceBook and Twitter page.
    Type: Marketing/Promotion


  4. Movie Night
    Purpose: In a social setting, show what types of content regarding biotechnology and social media are presented to the public. Demonstrate how that affects the overall perspective that they have towards the topic and how it shapes their future opinions.
    How: Show a very popular movie that has recently played or a well-known classic to the students at the University.
    Type: Awareness


  5. Basic Lab Training
    Community Brick
    Purpose: Since we are a co-operative education school (meaning many students within our University do 5 or more 4 month internships with employers) it helps to have a kick-start on basic lab skills and techniques to add onto their resumes when they may not have that experience yet. This will help to make them more competitive when applying for positions.
    How: Hold a one day training session where University students are taught basic skills used everyday in our wet lab.
    Type: Education


  6. Grade 11 Synthetic Biology Workshop
    Community Brick
    Purpose : Similar in nature to UW iGEM 2011 Outreach’s Grade 12 Synthetic Biology Workshop (you can find the Community Brick for this, this interactive workshop’s aim was to promote open discussion on industries and current issues within synthetic biology.
    How: Hold a workshop using lab space and the development of two new activities that would suit the Grade 11 Biology curriculum.
    Type: Education


  7. Tips and Tricks to Have a Successful Outreach Event
    Community Brick
    Purpose : To take what has been learnt throughout the year and streamline the key values into a valuable guide for those interested in pursuing outreach activities.
    How: Develop a brief information sheet that puts together key points for organizing outreach events.
    Type: Educational


IN DEPTH DESCRIPTION OF EVENTS:

  1. BIOTALKS OPEN PANEL DISCUSSION
    Community Brick (Communications Plan Plus Example)

    This event we are planning will be more than just a lecture series, but it is meant to be so much more. It is an experience, a place of discussion and intellectual exchange and of learning. Those are some of the visions we have for this event.

    By having key industry speakers do brief 20 minute lectures on topics such as: Clinical Trial Drug Development, Commercialization of Biomass and Energy Products and Entrepreneurial Barriers for Biotechnological Companies, we are hoping to bridge the level of discomfort scientists have when it comes to business. We want to encourage innovation amongst the highly technical individuals within our university and provide an open forum of educational resources for them to gain knowledge.

    Currently, UW iGEM has a core team of members working on this initiative and we are hoping to implement the BioTalks event in March 2013. We have completed our communications plan, gathered 3 speakers from the Pharmaceutical, Agricultural and Renewable Energy sectors and have separated the organizational team into three components: Director and Lead of Social Media Marketing (Ekta Bibra), Internal Communications and Logistics Lead (Anjali Arya) and Vision and Strategy Lead (Angela Biskupovic). Now we are working on meeting the appropriate potential sponsors and advisors.

    The creation of a communications plan is very helpful in creating clear-cut objectives and values, giving your team an over-arching mission and have stakeholders who you are proposing this event to understand exactly what you are trying to do. We have created a Community Brick that demonstrates a template for writing a Communications Plan as well as our BioTalks example that we are currently proposing to potential sponsors and advisors.

    Our next steps involve, the development of an aggressive marketing strategy through social media and direct marketing, raising funds and all the logistical work involved with a large-scale event such as this. Please stay tuned! If you have questions on this event feel free to contact us and we would love to share ideas with you!

    Contact the BioTalks team at: uwigem.outreach.hp@gmail.com (Ekta Bibra, Outreach Lead)

  2. ENGINEERING SCIENCE QUEST (ESQ)
  3. Community Brick Available for Protocol and Slide Deck

    Outreach took a part in the Engineering Science Quest at University of Waterloo by holding a lab activity for high school students (ages 14-17). The idea was to show students that it is easy to do Science at home.

    Gel Electrophoresis was performed using all supplies available at home (except for agarose- but one can use Jello too). We performed this activity with 3 different groups of ~10 students each time over the span of 2 months. We paired students together and gave them a chance to work in a team and learn some basic lab techniques such as pipetting, understanding the idea of having a medium for the electrons to flow in and much more.

    Students were also given a formal presentation at the end of the activity where the theory of Gel Electrophoresis was explained and were at the end asked a few questions for small prizes.

    Students were able to separate food dye (based on their charge) successfully; as 4 out of 5 groups, on average, did see a proper separation of colours.

  4. HOT CHOCOLATE EVENT


  5. The purpose of this event was to bring the UW campus community together and educate them about the field of synthetic biology through iGEM.

    The idea was to serve hot chocolate to individuals wishing to buy it for 50 cents a cup. While they are getting their drink served to them, iGEM members will be there engaging in conversations with the various students and faculty, discussing with them the history of synthetic biology and how it has evolved over the years.

    Hosting such an event especially a one that involves serving food to the local public, requires a lot of permission seeking for various personnels.

    The following protocol must be followed at UW in order to serve food publically anywhere on campus:

    • When serving food publically, one must first contact Feds and let them be aware of your ideas to host such event well in advance (estimated 4 weeks prior). Space is very limited on campus therefore letting Feds know of your plans somewhat secures your spot on campus for that day and time.

    • Next comes having the Waterloo Health Region approve your plans and your food before your serve them to the public. This is a fairly long process and usually teams/clubs/groups are advised to fill out this form and get in touch with Waterloo Health Region at least 8 weeks prior to your event.

    • While you are still waiting to hear from the Waterloo Health Region, make sure you keep in touch with what else is going on around campus that week that could possibly interfere with your event. For example I our case, UW’s Welcome Week BBQ lunch was being held at the same time same area, same day. Try and contact those individuals so you can work out the space and equipment (if sharing) so no issues arise on the day off the event.

    • In addition, have your ingredients (same ones listed on the WHN form) arranged and readily available on the day of your event.

    • Lastly, to save yourself time, if you will be using electronic equipments or large equipments such as water thermals that require you to bring hot water externally, have it arranged close to your location and already approved by the “company” supplying it to you.

    • Throughout this whole process keep in mind that communication is key. You will be in touch with various individuals from various groups and so be as descriptive and detailed as possible to get your point across.

  6. MOVIE NIGHT/ENTERTAINMENT NIGHT


  7. The purpose of this event was to show a movie related to Synthetic Biology in relation to what is new today (for a possible discussion later on).

    When starting an outreach event involving entertainment for the first time one must. Understand that the turnout may not be very high but remember it is a first-time event. Especially if it is held during midterm period.

    Next you must focus on getting in touch with Feds again and communicating with them to try and find a place where not only can you show a movie but also accommodate for the amount of people you expect to turn up for this event. This can be tricky if you have a very specific date picked out. Always have a range of dates as a backup plan because since space if limited on campus, you might not always get your desired location at the day and time you want it.

    If there are specific individuals in-charge of these events make sure they are aware of not only where to find the appropriate equipment needed to show the movie on campus but also the process involved in the rental. There are very strict protocols that need to be followed in regards to electrical equipment being rented at UW. Get in touch with media doc. for more details for UW.

    Another thing to keep in mind is that University of Waterloo is only allowed to show licensed movies/videos on campus. Feds is usually a great help in this process. They tend to have a huge list of movies available for you to choose from and in most cases the movie you want to show will be included in that licensed list. If not try and get in touch with one of the Feds executives or clubs manager to see if they can assist you in getting a license to show that movie on campus. The movie however will be rented from the closest store.

    One last thing to be kept in mind is, if your group plans on serving food at this event, make sure to follow the steps similar to the ones listed above for the food event.

  8. LABORATORY EVENT
  9. Community Brick Available for Protocol

    The purpose of this event was to educate science students about the basic and complicated lab techniques that are required during their undergraduate years here at Waterloo.

    This exercise followed a similar pattern to our ESQ exercises, however are made more directed towards first and second year students.

    Since this activity is very hands on, laboratory safety protocol is very important to follow. Each student that wants to participate in this activity must have their WHMIS certification before they show up for the event.

    For activities like this only a lab space can be used to perform the experiments. Therefore it is crucial that you get an instructor’s permission to his/her lab space. With regular lab sections utilizing the lab space for a minimum for 3 hours per section, time is very critical. In most cases this even will have to be held when a specific lab is over for the term.

    Details of the experiments being performed must be discussed by the instructor before anything is agreed upon. These details should include, the time and date of your event, what kind of experiments the students will be performing as well as what kind of equipment will be provided to them and if necessary what kind of equipment is required by the instructor of the lab to provide for us.

    Costs of the equipment and materials being used will also have to be calculated just in case there needs to be a charge placed on this.

    Keep in mind that lab spaces are not entirely meant for a large group of students. Prior to deciding on the exact date and time for your event, try and put out a survey to see how many students are actually interested. This will help you decide whether you should have one or two sessions and whether or not they should be on the same time and date or not.

    Before finalizing the details for the event just prepare a timeline and run it through the instructor as well as the volunteers teaching the students so everyone is on the same page.

    If someone within your team or outside of your team is making a commitment for the event that is critical for its execution, ensure that they either understand that once they make a commitment to a certain time they cannot leave or make them sign a contractual form binding them to the event. This will ensure no last minute cancellations.

  10. EXTERNAL WORKSHOPS
  11. Community Brick (Synthetic Biology and You, Gr.11)

    The purpose of this event is to education younger generation (Gr. 11 and 12 students) about the field of synthetic biology, what it involves and what kind of future it holds for them. The idea behind it is to let them be aware of the opportunities out there for them in the field of science.

    This workshop involves a selected science class from the local high school to come to campus for a whole day event. Being able to contact a High School teacher and letting them know about this opportunity for their students is the main part. It is slightly easier for the beginning year to keep it limited to local High Schools just because that way you are limiting any communication and travelling issues. Team members could also get in touch with their old high school science teacher to see if they are interested to attend.

    If the teacher is hearing about this for the first time it is usually beneficial to have brochures, pamphlets or postcards to distribute to the prospects. This makes sure that they leave with enough information to get them to start thinking about the idea of getting their students to come and learn about synthetic biology.

    Sometimes getting in touch with teachers can be hard. If you University/College has outreach programs that associate with high schools, get in touch with them, they can be a great aid in terms of networking with the High School teachers.

    Keep in mind that even if the High School is local, there needs to be a written contest of the teacher agreeing to perform this activity on the given date and any changes made should be informed to the other party in a well advanced notice. This should also be run through the school’s administration to avoid any last minute changes or cancellations. Set one effective deadline and aim to plan everything accordingly.

    In these types of activities communication between you group, the school and the teacher is essential.

    After those details have been finalized, similar room booking directions can be followed as listed before.

Motivation and Goals

This year's modelling project focused on extending the work done by the modelling team in 2010.


Waterloo's 2010 iGEM project, "Staphiscope", utilized amplifier parts developed by Cambridge in 2009 to detect low levels of Staph Aureus. These amplifier parts were characterized by the Cambridge team, but only under control of AraC/pBAD promoter, which differed from the promoter used in our 2010 Staphiscope project.


In order to characterize the amplifiers, a parameter scan was undertaken to find promoter-independent Hill parameters of each amplifier, consistent with data of full system. However, empirical verification of our results was lacking. This year, we sought to obtain this data, which (in conjunction with Cambridge data and model), would allow us to find Hill parameters for each amplifier.


Model

To allow for comparison of data, we used the same model as Cambridge in 2009.


In this model, araC represses the pBAD promoter in the absence of the inducer, arabinose. When arabinose is present, it binds to araC, preventing repression of the promoter and allowing transcription of reporter (GFP). This situation is modelled by a Hill function; we seek the Hill parameters of this function.


Thus, when AraC/pBAD system is induced with arabinose, we expect to see a steady increase of fluorescence from a low level, followed by a plateau of fluorescence at steady state.


Method

To measure fluorescence, we closely followed the assay described in the paper "Measuring the activity of BioBrick promoters using an in vivo reference standard", in the section "Assay of Promoter Collections".


Three cultures were grown overnight at 37 degrees Celsius with spinning at 200 rpm: untransformed BW27783, BW27783 containing BBa_I0500, and BW27783 containing BBa_I20260. These were then diluted 1:100 and regrown for roughly 4 hours under the same conditions. They were then diluted to an OD between 0.05 and 0.09, and regrown for 1 hour, again under the same conditions.


After this, the cultures were diluted into a 96-well plate at 8 different concentrations of inducer (arabinose), ranging from 0 to 6.4 uM. The plate was then incubated in a Wallac Victor3 multi-well fluorimeter at 37 degrees Celsius, and repeating measurements of absorbance and fluorescence were taken at 10 minute intervals, with shaking after each measurement. Untransformed BW27783, at each concentration of arabinose, was used to measure background fluorescence, and wells containing only broth were included to measure background absorbance. The machine settings used were identical to those described in the paper referenced above.


With this data, we aimed to calculate the steady-state per-cell GFP concentration during log-phase growth, for both BBa_I0500 and BBa_I20260 (measurement kit for the standard promoter, J23101). The ratio of these values would then characterize the strength of the AraC/pBAD promoter in units of RPU. The justification for this approach can be found in the supplemental material of the paper referenced above.


Results

The results of the experiment were anomalous, and considered too unreliable to be conclusive. There was no clear relationship between cell fluorescence and inducer concentration.


The fluorescence curve did not qualitatively match the predictions of the model; across all concentrations, and for each of the 3 cultures, we observed a high initial fluorescence, with a rapid drop to a lower steady state value. For each culture, this drop in fluorescence aligned well with the growth curve.


In addition, the untransformed BW27783 cells exhibited consistently higher fluorescence than cells containing BBa_I0500, which was highly anomalous. Because of this, we could not reliably use these cells to measure background fluorescence.


Below, a sample graph of Total Fluorescence is shown for each of the 3 cultures. These are curves of the total fluorescence for each culture, averaged over 3 replicates for each culture.


Discussion

It is believed that an error in our strain of BW27783 is most likely responsible for the anomalous qualitative features of our data. This is because for each concentration of inducer, the untransformed BW27783 cells exhibit a fluorescence curve highly similar to that of BW27783 containing BBa_I0500, and yet the untransformed cells should not be expressing GFP.


Prior to the measurement assay, BW27783 cells transformed with BBa_I0500 were plated and examined for fluorescence, both with and without the presence of inducer. The uninduced cells were not found to fluoresce, while the induced cells did fluoresce. The fluorescing cultures were used to make the frozen stock of BBa_I0500 which was used in the measurement assay. This indicates that our untransformed BW27783 should not fluoresce without the presence of inducer. Furthermore, the untransformed BW27783 cells used in the measurement assay were at no point prior to the assay exposed to arabinose.


To explain the fluorescence of the untransformed BW27783 in the measurement assay, it is speculated that our strain of BW27783 exhibits a rapid production of GFP in response to even low concentrations of inducer. Experimental error is also a likely source of inaccuracy in the data, although the qualitative features described were consistent across 3 trials of the experiment. Research into these results is still ongoing.


Determining The Factors Which Affect Perception of Synthetic Biology: A Multiple Regression Analysis


Despite synthetic biology's rapidly growing importance in a wide variety of fields including energy and health, it is still relatively unknown to the population at large. While some may have a vague notion of what synthetic biology is and its potential impact, most do not have anything to associate it with. In fact, some may even find the juxtaposition of artificial (synthetic) and natural (biology) confusing or contradictory. We at the iGEM University of Waterloo Human Practices team believe this represents a prime opportunity to help shape the public perception of synthetic biology and allay the fear and paranoia typically associated with the emergence of similar new fields of study. To do this effectively, we believe it's necessary to examine closely what factors or characteristics may affect a person's perception of synthetic biology. The purpose of this study, then, is to use statistical analysis, specifically regression modelling, to quantify these factors and their effect on perception. We created a survey to gather the data necessary for this analysis. It consists of three sections: first, background information to help identify the relevant factors for each respondent; next, a "pop" quiz designed to provide insight into the respondent's knowledge of synthetic biology; finally, a section that relates to the respondent's perception of synthetic biology and its uses.

The central question to be answered by this study is "what makes somebody more likely to have a favourable or unfavourable opinion of synthetic biology?" The purpose of this study is to determine the measurable effect of certain factors such as age and field of study or occupation on one's opinion of synthetic biology and its potential applications. This was to be accomplished via a regression model of the form yi = β0 + β1xi1 + β2xi2 +...+ βpxip + εi for i = 1, 2,...n,. Here y represents an individual's perception of synthetic biology, x represents each of the factors considered and β represents the corresponding quantifiable impact, positive or negative, of each factor on perception. Regression analysis was to be conducted using statistical software, most likely Stata or SPSS. The data needed for this analysis was to be collected via an online survey. Respondents will indicate the factors that correlate to them based on their answers to the questions in section #1 of the survey, while section #3 has been designed to reveal the respondent's current perception of synthetic biology. The second section of the survey is a short quiz to illustrate a respondent's level of knowledge and familiarity with synthetic biology. This factor is expected to be the major determinant in perception, along with age range and field of study/occupation. Other factors such as gender and geographic location within Ontario are expected to have no statistically significant impact on perception. The respondents were to come from a wide range of backgrounds in order to increase the robustness of our results.

The rationale behind this study is that by identifying the demographics that are most and least favourable toward synthetic biology and its expanding range of uses, the UW iGEM can more effectively target our efforts for raising awareness on the field. Despite synthetic biology's rapidly growing importance in a wide variety of fields including energy and health, it is still relatively unknown to the population at large. While some may have a vague notion of what synthetic biology is and its potential impact, most do not have anything to associate it with. In fact, some may even find the juxtaposition of artificial (synthetic) and natural (biology) confusing or contradictory. There are even organizations such as the ETC group that have published biased and one-sided reports ("Extreme Genetic Engineering: An Introduction to Synthetic Biology) that are threatening to greatly damage public opinion of synthetic biology. Biotechnology has faced a similar challenge as it has risen to prominence over the past two decades, with misinformation spread and the public lacking the fundamental knowledge necessary to critically interpret this information. In order to combat this reputation, we need to raise awareness of what synthetic biology is, along with an honest and unbiased assessment of its risks and benefits. As this can be a daunting task, the iGEM team decided to conduct this study as a way to help us focus our efforts and gain insight into the composition of perception.

After creating the survey questions, we distributed it online via Kwik Surveys using past co-op employers, campus clubs and other resources. Unfortunately we were not able to accrue enough responses to make any regression analysis statistically significant. Upon meeting with an econometrician in mid-September, we decided to refocus the survey on revealing the mechanism by which some groups end up with specific misconceptions regarding synthetic biology. As an (albeit very exaggerated) example, if the survey were to reveal that respondents with children were much more likely to support the notion that synthetic biology was "playing God by creating life," one might speculate that these respondents feel that as parents only they have exclusive domain of creating life. The value of this past year's project was to gain experience in the areas of survey creation and distribution, as well as to build connections with those who can help take next year's Human Practices to new levels.

TEST TEST page


Members
Advisors
About UW

OUR TEAM!

PeterPeter Hong
Director
Linda
Linda Yang
Assistant Director
Ekta
Ekta Bibra
Outreach Leader
Anjaji
Anjali Arya
Outreach Leader
Urooj
Urooj Kishor
Outreach Leader
Angela
Angela Biskupovic
Human Practices Leader
Simon
Simon Burru
Human Practices Leader
Aaron
Aaron Bender
Lab Project Leader
Artus
Dongbin Zhang
Lab Project Leader
Denise
Denise Lieuson
Lab Project Leader
Emily
Emily JunWen Li
Lab Project Leader
Rummy
Rummy Chowdhury
Lab Project Leader
KasiaKasia Karpinska-Leydier
Lab Team
AshleyAshley Ross
Lab Team
ReenaReena Paink
Lab Team
JordanJordan Lapointe
Mathematical Modelling Leader
Paul
Paul Reginato
Mathematical Modelling Leader
Kathryn
Kathryn Scannell
Mathematical Modelling Team

OUR ADVISORS!

Dr. IngallsDr. Brian Ingalls
Dr. CharlesDr. Trevor Charles
Dr. MoffattDr. Barb Moffatt
Dr. AucoinDr. Marc Aucoin

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.

Parts List

UW's parts for 2012


BBa_K576003 - RNA - Left part of self-excising ribozyme

BBa_K576004 - RNA - Right part of self-excising ribozyme

BBa_K576005 - Reporter - Left part of GFP (GFP 1) with promoter (J23101) and RBS (B0034)

BBa_K576006 - Reporter - Right part of GFP (GFP 2) with transcription terminator

BBa_K576007 - Intermediate - Left part of GFP with left part of self-excising ribozyme attached using RFC 53 construction.

BBa_K576008 - Intermediate - Right part of the self-excising ribozyme attached to the right part of GFP using RFC 53 construction

BBa_K576009 - Intermediate - Lox attached on to BBa_K576005 on the right of the part. Standard assembly (RFC 10) was used for this construction.

BBa_K576010 - Intermediate - Lox attached on to BBa_K576008 on the left of the part. BBa_K576009 or BBa_K576010 can be used depending on your convenience

BBa_K576011 - Reporter - Final construction of the 2011 project. The self-excising ribozyme should be cut out of from the rest of the sequence and thus expressing the full GFP.

BBa_K576012 - Reporter - Negative control of the experiment. The lox recombination site interrupts the GFP expression

BBa_K576013 - Reporter - Positive control of the experiment. Everything in between has been cut out by the self-excising intron and the GFP is fully expressed.


The UW 2012 lab project is a continuation from the 2011 one, thus no new parts are introduced.

Lab Work
Overview
Lab Journal
Entries
Lab Safety
N01
Fig 1. Construction map for experimental main construct
• K576005 contains the first component of GFP (GFPL)
• K576003 contains the first part of the intron sequence (INL)
• J61046 contains the lox site
• K576006 contains the second component of GFP (GFPR)
• K576004 contains the second part of the intron sequence (INR)
• K576007 contains GFPL and INL
• K576009 contains GFPL, INL and lox1
• K576011 contains the promoter (P), ribosomal binding sit (RBS), GFPL, INL, lox site, INR, GFPR and transcriptional terminator (TT). This is the final construct (experimental design)

N01
Fig 2. Construction map for positive and negative controls
• K576005 contains the first component of GFP (GFPL)
• K576006 contains the second component of GFP (GFPR)
• 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 (GFPL))
• J61046 contains the lox site
• K576006 contains the second component of GFP (GFPR)
• K576012 contains the promoter (P), ribosomal binding site (RBS), GFPL, lox site, GFPR and transcriptional terminator (TT). This is the negative control.

Lab book: Main construct

Week of June 17th-24th June 17th: First general meeting with lab volunteers, addressing general lab etiquettes and safety procedures. Explained the general lab working outline and project design. Exchanged contact information and constructed a lab schedule for volunteers and lab leaders.

June 18th: Lab leader (Emily) inoculated parts 118-PSB1C3 backbone(Cmr), 119-InL(Ampr), 120-InR(Ampr), 121-GFPR(Ampr),122-GFPL(Ampr) and 123-Lox (Ampr). All parts except 118 are in puc57, which is ampicillin resistant. Cultures inoculated in LB broth, incubated at 37° C overnight.

June 19th: Volunteers invited into the lab with a brief opening and orientation. Two volunteers trained and conducted miniprep of overnight cultures. Samples are quantified using Nanodrop Spectrophotometer

June 24th: Digested samples (118-122) with Fermentas Fast-digest restriction enzymes EcoR1 and Pst1 to subclone parts 119-122 into PSB1C3 (118). Digestion was incubated at 37° C for an hour (may be too long, but no star activities detected).

A diagnostic gel then follows by loading the cut samples along with the uncut minipreps for comparison.

129bp band observed for InL (119), 172bp observed for InR (120). 545bp and 399bps are observed for GFPL (122) and GFPR (121) respectively. Differences observed between the undigested and digested samples.

June 27th: Run the sample gel as last time but with no control. Gel extracted samples using Biobasic and fermentas binding buffers.

June 28th: Nanodrop results show very low concentration (less than 1uL) for InL and 2. Gel extraction need to be repeated

June 29th: Gel extraction repeated and concentration greatly increased. Enough for ligation

July 4th: Ligation using 1:3 vector to insert ratio in moles. InL, InR, GFPL and GFPR are ligated into PSB1C3 with vector+ ligase control and vector only control. The ligation tubes are incubated overnight at room temperature.

July 5th: Transformation into DH5a cells using heat shock technique. Cells plated on LB Cmr plates and incubated overnight at 37° C. Added transformation control (vector uncut into DH5a) and negative control (DH5a cells only). 1/10 and 9/10 dilutions for each ligation

July 6th: Took out transformation plates. Pink colonies (hundreds) only observed on vector+ligase, vector only controls and transformation controls. Negative control plates are clean. Plates with insert and plasmid have hundreds of white colonies and some pink colonies present. Two morphologies present; large and small

July 9th: Streak purified 24 white colonies selectively (6 of each 3 large, 3 small)

July 10: Inoculate and patched colonies into LB CmR tubes/plates

July 11: Picked 8 final colonies to screen (1 big and 1 small each set) Miniprep these samples

July 12: Digested samples to check for insert (with EcoR1 and Pst1). Loaded a diagnostic gel afterwards

Result: Proper sized inserts were observed for all samples. All parts were subcloned successfully into PSB1C3

July 13: Inoculated for more miniprep

July 14: Miniprep samples again and saved frozen stock of samples

July 18th: Digestion of InL and GFPL with BgIII and EarI (sites overlap, put EarI in first and add BgIII after 30 minutes). Insert (115bp) observed for digestion with InL

July 19th: Gel extracted InL and GFPL

July 21st: Ligated InL with GFPL with 1:3 insert: vector ratio in moles. Incubated at room temperature overnight

July 23rd: Transformed ligation product with transformation and negative control on LB Cm plates

July 24th: All cells had a lawn of cells.

July 26th: Miniprep and digested using the same protocol as above

July 27thh: Miniprep PSB1C3

July 28th: Miniprep lox and digested lox using Xbal and Pst1. Digested lox loaded onto 2% gel.

August 2nd: Gel extracted InL and GFPL again for ligation and conducted ligation again

August 9th: Transformation into DH5a

August 13: Repeat miniprep of InL and GFPL

August 14th: Digested InL and GFPL with EarI and BgIII again

Exam break

August 28th: Final ligation of InL and GFPL

August 29th: Transformation. All colonies are white, but more colonies present for the inesrt than vector + ligase only.

September 17: Picked colonies for inoculation

September 18: Miniprep of samples

September 19: Digestion of samples and loading of gel. No inserts present.

Lab book: Controls

June 21st 2012: Enzyme digestion and gel extraction of pSB1C3, BBa_K576005 in pUC57, BBa_K576006 in pUC57 for subcloning. There were unexpected bands on the gel we will have to redo experiment.

June 29th 2012: Enzyme digestion and gel extraction of pSB1C3, BBa_K576005 and BBa_K576006 for subcloning.

July 7th 2012: Ligation of BBa_K576005 into pSB1C3 and ligation of BBa_K576006 into pSB1C3 for subcloning.

July 10th 2012: Transformation of BBa_K576005 in pSB1C3 and BBa_K576006 in pSB1C3 into DH5-alpha for subcloning.

July 11th 2012: Checked the plates and they were all lawns. We will use Emilys subcloned parts for now.

July 17th 2012: Inoculation of pSB1C3 containing strain and BBa_J61046 in pUC57 containing strain.

July 18th 2012: Our frozen stock may be contaminated so we discarded the inoculation tubes from July 17th and we will use Emilys miniprepped samples for now. Restriction digest and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006.

July 21st 2012: Ligation to make BBa_K576013 construct.

July 22nd 2012: Transformation of BBa_K576013 construct into DH5-alpha.

July 23rd 2012: All the plates were lawns. Shaking the broth we used showed it was contaminated. Discard the plates and repeat the experiment.

July 28th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. The bands on the gel looked as expected but the nanodrop values after the gel extraction were indicated we did not have DNA. We will repeat the digest and gel extraction using isopropanol to increase yield.

August 18th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Ligation to make BBa_K576013 construct.

August 19th 2012: Transformation of BBa_K576013 construct into DH5-alpha. Transformation of left construct intermediate for the main construct.

August 20th 2012: All the plates were lawns. This time we are confident the broth was not contaminated. Our problem could be with the antibiotic. Next time we will plate a 1/10 dilution of the cells.

August 21st 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. The bands on the gel looked as expected but the nanodrop values after the gel extraction were not good enough to move onto ligation.

August 26th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Restriction digestion of pSB1C5. pSB1C5 showed an unexpected band in the gel so we did not proceed to gel extraction.

August 28th 2012: Ligation to make BBa_K576013 construct.

August 29th 2012: Transformation of BBa_K576013 construct into DH5-alpha. Transformation of left construct intermediate for main construct into DH5-alpha.

August 30th 2012: All the plates were clean except the transformation of the miniprepped sample for a positive control had colonies. Our problem is therefore not with the transformation. Our problem may be with our ligation. We will repeat the experiment with an additional control for the ligation.

September 6th 2012: Restriction digestion and gel extraction of BBa_K576005 in pSB1C3 and BBa_K576006. Linearize and gel extract pSB1C3 for additional ligation control. Ligation to make BBa_K576013 construct and ligation of linearized pSB1C3.

September 7th 2012: Transformation of BBa_K576013 construct into DH5-alpha.

September 8th 2012: The transformed miniprep and transformed ligated pSB1C3 had colonies. The rest of the plates were clean. Therefore the transformation and ligation was successful however the construct still didn't work out. We will repeat the experiment after miniprepping more BBa_K576006 in pSB1C3. Inoculate BBa_K576005 in pSB1C3.

September 9th 2012: Miniprep BBa_K576006 in pSB1C3.

September 11th 2012: Streak purify DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576005 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 from frozen stock.

September 12th 2012: Inoculate DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576005 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 in Terrific broth.

September 13th 2012: Miniprep BBa_K576005 in pSB1C3, BBa_K576006 in pSB1C3 and BBa_J61046 in pUC57.

September 15th 2012: Restriction digestion of parts BBa_K576005, BBa_K576006 and BBa_J61046 to get gel extracted fragments for both the positive and negative control constructs. Diagnostic gel showed questionable bands so we need to repeat the digestion.

September 16th 2012: Repeat the digestion from September 15th. This time the bands are as expected so the fragments were gel extracted. The nanodrop values of gel extracted BBa_K576005 in pSB1C3 for the negative control construct indicated we did not have DNA so need to redo that sample. Other samples were stored.

September 18th 2012: Restriction digestion of BBa_K576005 in pSB1C3 for negative control construct but the gel showed an unexpected band so did not gel extract. Ligation to make BBa_K576013 construct. Inoculation of DH5-alpha carrying BBa_K576005 in pSB1C3, DH5-alpha carrying BBa_K576006 in pSB1C3, DH5-alpha carrying BBa_K576003 in pSB1C3, DH5-alpha carrying BBa_K576004 in pSB1C3 and DH5-alpha carrying BBa_J61046 in pUC57 in Terrific broth.

September 19th 2012: Miniprep of BBa_K576005 in pSB1C3, BBa_K576006 in pSB1C3, BBa_K576003 in pSB1C3, BBa_K576004 in pSB1C3 and BBa_J61046 in pUC57. Transformation of BBa_K576013 construct into DH5-alpha.

September 20th 2012: All plates were clean except the transformed miniprep. Stop lab work on BBa_K576013 construct due to time constraints.

September 23rd 2012: Restriction digestion of minipreps from 1-12 colonies to screen left construct transformation plate. Load diagnostic amounts of digested samples to image BBa_K576005, BBa_K576006, BBa_K576003, BBa_K576004 and BBa_J61046 bands in a gel to demonstrate successful subcloning. The BBa_K576003 and BBa_K576004 bands were not visible in the gel.

September 24th 2012: Load diagnostic amounts of digested miniprepped 1-10 colonies to screen left construct plate.

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