Team:Minnesota/Outreach/ECORI

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<h1>Middle School Curriculum</h1><br>
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<p text-align:left>
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<a href="https://static.igem.org/mediawiki/2013/4/44/GopheriGEM_ECORI_Squad_2Day_Synbio_Curriculum.pdf">Click here for our curriculum!</a>
 +
</p>
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<iframe src="http://prezi.com/embed/fasbbvuqb7ux/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"></iframe>  
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<h1>Designing a BioBrick Compatible Pichia Expression System</h1><br>
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&nbsp;&nbsp;&nbsp; The ECORI Squad developed a curriculum to teach the community about synthetic biology, research innovation, and bioethics. To test if the lesson plans would be effective, ECORI visited Salk Middle School in Elk River, Minnesota, taught five classes and reached out to over 150 students. The lesson plan consisted of two days of hands-on experiments, coupled with brief interactive lectures.<br>
 +
<br>
 +
&nbsp;&nbsp;&nbsp;The first day, the ECORI Squad introduced students to synthetic biology. The initial activity we instructed students on how to extract DNA from strawberries using household items. Next, the students were able to experience making art with colorful modified organisms by plating bacteria on agar plates in any design of their choosing. The bacterial organism we used were bioluminescent, fluorescent green, fluorescent red, and blue (particular strains can be found in our curriculum handbook [have handbook link out to PDF of synbio curriculum.)  We explained to students proper protocol for handling and disposing of genetically modified organisms, along with proper aseptic technique, and lab safety procedures.  We then brought students together to conclude the curriculum for Day 1.<br>
 +
<br>
 +
&nbsp;&nbsp;&nbsp;Our second day at Salk Middle School, students were radiating with excitement (much like the bacterial plates they would view later on!) We started the day with a brief lecture on restriction enzymes, and how they are used to manipulate DNA. We introduced the concept of BioBricking plasmids, and let the students learn hands on how to construct different vectors from plasmid components. This activity was an exercise of logic, and the students grasped the concept very readily! After the students correctly designed the target vector, they were asked what functions they inserted into their bacterial plasmids. Some examples produced by the students were:<br>
 +
<ul>
 +
  <li>Do my homework!</li>
 +
  <li>Eat trash, while still smelling good</li>
 +
  <li>Drive a Lamborghini</li>
 +
  <li>Target “sick cells” and make them glow, making them easier to detect</li>
 +
  <li>Play videogames!</li>
 +
  <li>Make clothes</li>
 +
</ul>
 +
<br><br>
 +
&nbsp;&nbsp;&nbsp;We explained that some of the ideas are far-fetched, but some of the vector designs that they came up with are actual research projects that are being worked on at the University of Minnesota! The students were very innovative in their thought process, and demonstrated how much they had learned in their two days of lecture. Below is a collage of drawings we collected from students prior to having any lecture, and an after picture we collected after
 +
the conclusion of their 100 minutes of learning about synthetic biology.<br><br>
 +
&nbsp;&nbsp;&nbsp;Finally, we concluded the session with a Q and A in which students could ask us whatever they wanted about synthetic biology. We addressed questions proposed by students concerning human trials, or potential environment issues. We answered these questions by engaging students in an ethical conversation. We had originally developed a discussion session exclusively regarding bioethics, but deemed it inappropriate for the level of philosophy taught in middle schools.<br>
 +
In order for this curriculum to suit an older age group, the ethics portion would contain discussion about what we can innovate using synthetic biology, and what would be better left alone. We would engage students with the following questions:<br><br>
 +
<ul>
 +
  <li>Where should we draw the line concerning manipulating human genetics?
 +
  <li>How would modified organisms be responsibly controlled?
 +
  <li>What would be the impact of introducing genetically altered organisms into a preexisting ecosystem?
 +
</ul>
<br>
<br>
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<center><font size="3"><b>Basic BioBrick Expression System Background </b></font></center>
 
<br>
<br>
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<p>
 
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<i><u><font size="4">• </font>What's the idea behind this expression system?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; Laboratory and industrial projects that involve the high volume production of a protein often select E. coli as an expression system due to its rapid growth. However, bacterial expression systems are not always a viable option. In the case where proper folding of the protein of interest requires post-translational modification (such as the addition of disulfide bonds or glycosylation,) a eukaryote must be used. Although several well-defined eukaryotic options exist, yeast is often selected for its ease of use in the laboratory. One yeast species in particular, Pichia pastoris, has gained popularity as an expression system for recombinant human proteins.
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font>Why did we choose P. Pastoris?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; P. pastoris has a glycosylation pattern that is more compatible with the human immune system, when compared to the glycosylation pattern of Saccharomyces cerevisiae. P. pastoris is also known for its ability to grow in high densities using methanol as its only food source. Despite the usefulness of yeast species such as P. pastoris there are currently few items in the parts registry that are designed for use within yeast, and none that are specifically designed to be used with P. pastoris. Our team intends on producing pBB3G1 and pBB1Z1, two BioBrick compatible P. pastoris-E. coli shuttle vectors.
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font>What are some benefits of this vector system?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; The pBB3G1 and pBB1Z1 vectors include features that increase its ease-of-use and versatility, such as: optional inducibility, optional product secretion, trans-kingdom conjugation (TKC), and episomal maintenance of the vector in the host organism. The vectors vary in their expression level. Constitutive expression is achieved in pBB1Z1 by the pGAP promoter. Methanol-induced expression is available in pBB3G1 by means of the pAOX1 promoter.
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font> Why use Trans Kingdom Conjugation instead of traditional transformation methods?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; TKC involves the transfer of DNA from a bacterial cell to a eukaryotic cell, by means of conjugation. Harnessing the ability to shuttle plasmids between E. coli and P. pastoris through TKC would simplify transformation protocols. Currently, transformation methods (using shuttle plasmids cloned in E. coli) are a time consuming process, requiring isolation of the cloned plasmid and transformation into yeast. Transformation using TKC shortens the process by transferring the plasmid directly into the yeast cell. Utilizing TKC as a transformation protocol would translate to faster results in the laboratory and reduced costs in an industrial setting. Currently there are no BioBrick vectors in the parts registry that enable TKC between E. coli and P. pastoris.
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font>How will TKC be achieved?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; TKC functionality is provided by the OriT<sup>P</sup> sequence which hosts the nick site that will be cleaved during the initiation of conjugation in order to linearize the plasmid, and ligated once transferred to the recipient. Importantly, the OriT<sup>P</sup> sequence -compared to other variations of OriT- does not require the presence of a helper plasmid within the recipient to complete the final ligation step of conjugal transfer. 
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font>Are there limitations to this system?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; One limitation to using P. pastoris as an expression system is that most shuttle plasmids must be integrated into the yeast chromosome. This results in lower expression of desired protein products, as well as lower transformation efficiency. We hope to improve the functionality of the pBB3G1/pBB1Z1 system by allowing the plasmid -once transferred to the yeast cell- to remain as an episomal plasmid. This is made possible by the inclusion of the PARS1 yeast autonomous replication sequence. This sequence ensures that the plasmid is maintained through several (~200) generations.
 
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<br>
 
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<br>
 
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<i><u><font size="4">• </font>How will we screen the genes?</u></i>
 
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<br>
 
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&nbsp; &nbsp; &nbsp; Transformant selection is simplified by the inclusion of antibiotic resistance genes that are effective in both E. coli and P. pastoris. The backbones that will be submitted to the parts registry will include either Zeocin or Geneticin, however the resistance genes have been cloned into a KpnI site, and may be swapped for any selective marker specific to the user’s needs.
 
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</p>
 
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<p><b>Goal</b><br>  To design a BioBrick compatible <i>E. coli</i>-<i>P. pastoris</i> shuttle vector.</p>
 
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<p><b>Background</b><br>
 
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</p>
 
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<!--2013 edit <img style="width:500px;" src="http://i1158.photobucket.com/albums/p607/iGEM_MN/caffeine_synth_resize.png">
 
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<p><b>Figure 1.  Scheme showing proposed caffeine biosynthetic pathway in S. cerevisiae. </b></p>
 
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<p><b>Methods</b><br>
 
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<b>Vector Design</b><br>
 
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&nbsp;&nbsp;&nbsp;Sequences for AOX1, GAP, G418, and Zeocin were obtained from pPICZ, pPICZα, pGAP from Invitrogen. The Wintergreen odor generator (BBa_J45700) was selected as a reporter gene, as well as yeast green fluorescent protein (GFP). In order to improve upon an existing part in the Registry of Standard Biological Parts, we codon-optimized the Wintergreen odor generator for expression in P. pastoris using the Codon Optimization tool provided by IDT. Kozak sequences were added for each open reading frame to be expressed in P. pastoris. The basal vectors pMNBB-ICI and pMNBB-CCI (please refer to Vector Nomenclature, and Figure 1) were assembled from the above sequences in Clone Manager 9.
 
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<!-- 2013 edit
 
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After settling on the two genes, the sequences were taken from NCBI and finalized for synthetic synthesis (XMT1 accession number DQ422954 and DXMT1 accession number DQ422955). A program was designed to optimize these plant genes for yeast use. Because of the similarity of the two genes (~88% similarity) the program was also designed to identify regions of homology between the two genes and adjust the codon usage such that homologous recombination will not occur at the nucleotide level while the overall protein sequence would be retained. The codons were chosen in descending order of complementary tRNA abundance. Any BioBrick cut sites found within the gene sequences were also removed.</p>
 
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<p>To synthesize the overall plasmid backbone, PCR primers were designed to amplify five desired fragments (with 25-30 bp overlap) from different plasmids, which could be joined by Gibson Assembly:<br>
 
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1.&nbsp;&nbsp;BioBrick destination plasmid pSB1C3 containing the MCS and rep (pMB1).<br>
 
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2.&nbsp;&nbsp;BioBrick destination plasmid pSB1C3 containing the Chloramphenical resistance gene. <br>
 
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3.&nbsp;&nbsp;2u Gibson Extract provided by the Schmidt-Dannert lab (University of Minnesota) containing the&nbsp;&nbsp;2u ORI for yeast. <br>
 
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4.&nbsp;&nbsp;pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1.<br>
 
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5.&nbsp;&nbsp;ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the &nbsp;&nbsp;&nbsp;&nbsp;G418 R gene.</p>
 
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<p><b>Parts List</b><br>
 
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<!-- 2013 edit
 
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BBa_K814000&nbsp;&nbsp;    dehydroquinate synthase (DHQS) generator<br>
 
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BBa_K814001&nbsp;&nbsp;    ATP-grasp (ATPG) generator<br>
 
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BBa_K814002&nbsp;&nbsp;    dehydroquinate O-methyltrasferase (O-MT) generator<br>
 
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BBa_K814003&nbsp;&nbsp;    shinorine non-ribosomal peptide synthase (NRPS) generator<br>
 
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BBa_K814004&nbsp;&nbsp;    mycosporine-glycine biosynthetic pathway<br>
 
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BBa_K814005&nbsp;&nbsp;    shinorine biosynthetic pathway<br>
 
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BBa_K814006&nbsp;&nbsp;    negative control for mycosporine-like amino acid biosynthesis<br>
 
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BBa_K814007&nbsp;&nbsp;    ScyA (acetolactate synthase) generator<br>
 
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BBa_K814008&nbsp;&nbsp;    ScyB (leucine dehydrogenase) generator<br>
 
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BBa_K814009&nbsp;&nbsp;    ScyC generator<br>
 
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BBa_K814010&nbsp;&nbsp;    partial scytonemin biosynthetic pathway, scyCB<br>
 
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BBa_K814011&nbsp;&nbsp;    scytonemin biosynthetic pathway, scyBAC<br>
 
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BBa_K814012&nbsp;&nbsp;    XMT1 protein generator<br>
 
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BBa_K814013&nbsp;&nbsp;    DXMT1 protein generator
 
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</p>
 
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<p><b>Figure 2:</b>  Pieces for Gibson assembly of our novel, shuttle backbone.  The individual pieces relate to the described components enumerated above. 1.  Purple arrow; 2.  Orange arrow; 3.  Green arrow; 4.  Blue arrow; 5.  Light green arrow.</p>
 
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/cells.jpg
 
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">
 
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<p><b>Figure 3. Creation of a shuttle vector designed for BioBrick components.</b>  A), Overnight culture of the pSB1C3 shipping vector showing RFP expression (a blank LB tube is shown as a control on the right).  B), RFP expression in the pGHMM2012 shuttle vector.  Cell pellets clearly indicate expression of RFP from this plasmid.  C), PCR screening for caffeine biosynthetic components into pGHMM2012.  The panels from left to right are positive clones for Xmt, Dxmt and controls for each of these components.</p>
 
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<img style="width:500px;" src="http://i1158.photobucket.com/albums/p607/iGEM_MN/growth_curve_resize.png">
 
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<p><b>Figure 4. Investigation of caffeine toxicity in yeast. </b>The yeast were resilient in the presence of caffeine and there was no significant decrease in growth overtime at each different concentration.  For reference, the concentration of caffeine in coffee is roughly 600mg/L.</p>
 
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<img src="http://i1158.photobucket.com/albums/p607/iGEM_MN/hplccaffeine-1_resize.jpg">
 
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<p><b>Figure 5. HPLC Data. </b>description.</p>
 
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<p><b>Conclusions</b><br>
 
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We were able to design and synthesize a novel yeast- E. coli hybrid plasmid which was optimized to prevent homologous recombination in our two synthetic genes: XMT1 and DXMT1. </p>
 
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<p>If the HPLC results can confirm the presence of caffeine in the yeast cultures, we would confirm the presence of the two target proteins by SDS-PAGE, possibly even performing antibody detection if there is difficulty confirming the presence.</p>
 
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<p>If the HPLC results cannot confirm the presence of caffeine, the first step would be to sequence our genes and align them with the sequences submitted to IDT for the gBlocks. We could also measure the production of the intermediates compared to the cultures to test whether caffeine synthesis is being arrested in the pathway.</p>
 
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Latest revision as of 19:11, 29 November 2013

Team:Minnesota - Main Style Template Team:Minnesota - Template







Middle School Curriculum


Click here for our curriculum!

    The ECORI Squad developed a curriculum to teach the community about synthetic biology, research innovation, and bioethics. To test if the lesson plans would be effective, ECORI visited Salk Middle School in Elk River, Minnesota, taught five classes and reached out to over 150 students. The lesson plan consisted of two days of hands-on experiments, coupled with brief interactive lectures.

   The first day, the ECORI Squad introduced students to synthetic biology. The initial activity we instructed students on how to extract DNA from strawberries using household items. Next, the students were able to experience making art with colorful modified organisms by plating bacteria on agar plates in any design of their choosing. The bacterial organism we used were bioluminescent, fluorescent green, fluorescent red, and blue (particular strains can be found in our curriculum handbook [have handbook link out to PDF of synbio curriculum.) We explained to students proper protocol for handling and disposing of genetically modified organisms, along with proper aseptic technique, and lab safety procedures. We then brought students together to conclude the curriculum for Day 1.

   Our second day at Salk Middle School, students were radiating with excitement (much like the bacterial plates they would view later on!) We started the day with a brief lecture on restriction enzymes, and how they are used to manipulate DNA. We introduced the concept of BioBricking plasmids, and let the students learn hands on how to construct different vectors from plasmid components. This activity was an exercise of logic, and the students grasped the concept very readily! After the students correctly designed the target vector, they were asked what functions they inserted into their bacterial plasmids. Some examples produced by the students were:
  • Do my homework!
  • Eat trash, while still smelling good
  • Drive a Lamborghini
  • Target “sick cells” and make them glow, making them easier to detect
  • Play videogames!
  • Make clothes


   We explained that some of the ideas are far-fetched, but some of the vector designs that they came up with are actual research projects that are being worked on at the University of Minnesota! The students were very innovative in their thought process, and demonstrated how much they had learned in their two days of lecture. Below is a collage of drawings we collected from students prior to having any lecture, and an after picture we collected after the conclusion of their 100 minutes of learning about synthetic biology.

   Finally, we concluded the session with a Q and A in which students could ask us whatever they wanted about synthetic biology. We addressed questions proposed by students concerning human trials, or potential environment issues. We answered these questions by engaging students in an ethical conversation. We had originally developed a discussion session exclusively regarding bioethics, but deemed it inappropriate for the level of philosophy taught in middle schools.
In order for this curriculum to suit an older age group, the ethics portion would contain discussion about what we can innovate using synthetic biology, and what would be better left alone. We would engage students with the following questions:

  • Where should we draw the line concerning manipulating human genetics?
  • How would modified organisms be responsibly controlled?
  • What would be the impact of introducing genetically altered organisms into a preexisting ecosystem?