Team:MiamiU OH/Contribution

iGEM 2021 | Miami University

Contributions

Cyanobacteria in the Registry

We created new parts for future iGEM teams choosing to work with cyanobacteria, particularly our model organism Synechococcus elongatus PCC 7942. This model organism is ideal for studying photosynthesis due to its well characterized metabolic pathways and natural competency that readily allows transformation. Cyanobacteria are relatively low-cost and easy to maintain, as well as extremely safe experimental organisms as highlighted on our Safety page. Overall, these features make cyanobacteria an excellent choice as a model or chassis, especially in synthetic biology.

The iGEM Registry currently has few parts specific for cyanobacteria, contrasting with the well characterized parts for Escherichia coli and Saccharomyces cerevisiae. This lack of available constructs for cyanobacteria limits future photosynthetic-focused iGEM work, as these heterotrophic organism parts are usually not as effective or applicable when applied to cyanobacteria. This shortage requires major expansion, reorganization, and characterization of parts and tools to expand future iGEM opportunities and photosynthetic research.

As part of our project, we created three overexpression plasmids and one deletion plasmid, detailed below. Within these plasmids, consisting of several composite parts, we added numerous basic parts involving regulatory and expression functions within cyanobacteria. All our registered parts are detailed below in contextual descriptions, with technical details being on the linked registry pages.

Parts Registered

Overexpression Plasmids

Our overexpression plasmids were created via Gibson Assembly, of which we registered all relevant parts. These plasmids were all used within Synechococcus elongatus PCC 7942 with intended alterations to the Calvin-Bensen-Bassham (CBB) cycle.

BBa_K4047000 and BBa_K4047001 are primers for amplifying the transaldolase (tal) gene (BBa_K4047034) from wild type S. elongatus PCC 7942. The primer contains EcoRI and BamHI restriction enzyme sites for confirmation testing. Transaldolase participates in the CBB cycle by catalyzing the forward and reversion conversion of sedoheptulose-1,7-bisphosphate and glyceraldehyde-3-phosphate into erythrose-4-phosphate and fructose-1,6-bisphosphate. In a similar fashion, BBa_K4047002 and BBa_K4047003 are primers for amplification of the fructose-1,6-bisphosphatase (fbp) gene (BBa_K4047035). They also contained the EcoRI and BamHI restriction enzyme sites. Fructose-1,6-bisphosphatase catalyzes the hydrolysis of a phosphate group from fructose-1,6-bisphosphate. This process, part of the CBB cycle, is also vital to many other central metabolic pathways.

Additional primers were designed so that both tal and fbp, once amplified, could be inserted in a third overexpression plasmid (BBa_K4047004 and BBa_K4047005).

All our overexpression plasmids used pAM2991 as the expression vector (BBa_K4047024). This vector was created by Susan Golden in 2005 specifically for S. elongatus PCC 7942, and is publically available on addgene.org. Its multiple components include an origin of replication (BBa_K4047029), a trc promoter (BBa_K4047032), an rrnB T1 terminator (BBa_K4047025), an rrnB T2 terminator (BBa_K4047026), a base of mobility region (BBa_K4047028), a lac repressor (BBa_K4047031), a lacIq promoter (BBa_K4047030), a lac operator (BBa_K4047033), and spectinomycin/streptomycin resistance (BBa_K4047027). Sequences homologous to the neutral site I region allow for these components along with our target genes to be inserted into the genome of S. elongatus PCC 7942.

Our first plasmid, called pGEM-tal (BBa_K4047036), aimed to induce transaldolase overexpression by inserting an additional copy of the encoding gene, tal (Figure 1).

Annotated SnapGene viewer of pGEM-tal.Figure 1: Annotated SnapGene viewer of pGEM-tal.

Our second plasmid, named pGEM-fbp (BBa_K4047037), induced fructose-1,6-bisphosphate overexpression by inserting an additional copy of the encoding fbp gene (Figure 2).

Annotated SnapGene viewer of pGEM-fbp.Figure 2: Annotated SnapGene viewer of pGEM-fbp.

Finally, our third overexpression plasmid, pGEM-tal+fbp (BBa_K4047038), combined the encoding elements of the above overexpression plasmids, aiming to insert both tal and fbp genes to overexpress both genes (Figure 3).

Annotated SnapGene viewer of pGEM-tal+fbp.Figure 3: Annotated SnapGene viewer of pGEM-tal+fbp.

Deletion Plasmid

Our deletion plasmid, named pGEM-SBPase (BBa_K4047039), was designed to delete sedoheptulose-1,7-bisphosphate (glpX) from the S. elongatus PCC 7942 genome through homologous recombination with upstream (BBa_K4047022) and downstream (BBa_K4047023) sequences (Figure 4). Two sets of primers (BBa_K4047009 and BBa_K40470010, BBa_K4047013 and BBa_K4047014) were used to amplify 800 base pair upstream and downstream fragments of glpX from the S. elongatus genome. We designed additional primers (BBa_K4047007 and Bba_K4047008) to amplify the backbone of pXWK3-glgC. This backbone is well-established as a vector within the Wang Microbiology Lab at Miami University, used in a previous publication. The backbone (BBa_K4047018) contains an origin of replication and a gentamicin resistance gene (BBa_K4047016), promoter (BBa_K4047015), and terminator (BBa_K4047017). In addition, we used primers (BBa_K4047011 and BBa_K4047012) to amplify a kanamycin resistance cassette from pXWK3-glgC (BBa_K4047019, BBa_K4047020, BBa_K4047021).

Figure 4: Annotated SnapGene viewer of pGEM-SBPase.

Part compatibility

Our parts have a variety of compatibility, with the majority of our parts being compatible with all RFC standards. All of our plasmids, due to possessing various restriction sites, are not RFC compatible. However, these plasmids and several of their components, despite having limited RFC compatibility, may be extremely useful for future cyanobacteria teams due to their vast array of functions, suitability for the widely adaptable and accessible pAM2991 vector, and handling of the central metabolic genes tal and fbp.

Table 1: Complete list of registered parts from the MiamiU_OH team.

Name Type Description Designer Length
    W BBa_K4047016 Coding Gentamicin resistance (GmR) for Synechococcus An Nguyen 534
    W BBa_K4047019 Coding Kanamycin Resistance for Synechococcus An Nguyen 816
    W BBa_K4047020 Regulatory Kanamycin Promoter for Synechococcus An Nguyen 106
    W BBa_K4047021 Terminator Kanamycin Resistance Terminator for Synechococcus An Nguyen 311
    W BBa_K4047031 Regulatory lac repressor An Nguyen 1083
    W BBa_K4047034 Coding transaldolase An Nguyen 1194
U    BBa_K4047000 Primer primerT/TF_tal- forward Hope Kirby, Avery Imes 47
U    BBa_K4047001 Primer primerT_tal-reverse An Nguyen, Avery Imes 43
U    BBa_K4047002 Primer primerF_fbp-foward An Nguyen, Avery Imes 45
U    BBa_K4047003 Primer primerF/TF_fbp-reverse An Nguyen, Avery Imes 43
U    BBa_K4047004 Primer primerTF_tal-reverse An Nguyen, Avery Imes 38
U    BBa_K4047005 Primer primerTF_fbp-foward An Nguyen, Avery Imes 40
U    BBa_K4047007 Primer dSBPase_Backbone-fwd An Nguyen 37
U    BBa_K4047008 Primer dSBPase_Backbone-rev An Nguyen 18
U    BBa_K4047009 Primer dSBPase_glpX_US-fwd An Nguyen 41
U    BBa_K4047010 Primer dSBPase_glpX_US-rev An Nguyen 28
U    BBa_K4047011 Primer dSBPase_KanR-fwd An Nguyen 28
U    BBa_K4047012 Primer dSBPase_KanR-rev An Nguyen 34
U    BBa_K4047013 Primer dSBPase_glpX_DS-fwd An Nguyen 30
U    BBa_K4047014 Primer dSBPase_glpX_DS-rev An Nguyen 40
  W BBa_K4047015 Regulatory dSBPase-Backbonecomponent-GmRPromoter An Nguyen 316
  W BBa_K4047017 Terminator dSBPase-Backbone-GmrTerm An Nguyen 36
  W BBa_K4047018 Plasmid_Backbone dSBPase-Backbone An Nguyen 1781
    BBa_K4047022 DNA glpX_US An Nguyen 801
    BBa_K4047023 DNA glpX_DS An Nguyen 801
  W BBa_K4047024 Plasmid_Backbone pAM2991 An Nguyen 7973
    BBa_K4047025 Terminator rrnB T1 terminator An Nguyen 87
    BBa_K4047026 Terminator rrnB T2 terminoator An Nguyen 28
  W BBa_K4047027 Reporter SmR An Nguyen 792
     BBa_K4047028 Plasmid_Backbone bom An Nguyen 141
     BBa_K4047029 Plasmid_Backbone ori An Nguyen 589
    BBa_K4047030 Regulatory lacIq promoter An Nguyen 78
  W BBa_K4047032 Regulatory Trc promoter An Nguyen 30
     BBa_K4047033 Regulatory Lac operator An Nguyen 17
  W BBa_K4047035 Coding Fructose-1,6-bisphosphatase An Nguyen 1035
  W BBa_K4047036 Plasmid pGEM-tal An Nguyen 9172
  W BBa_K4047037 Plasmid pGEM-fbp An Nguyen 9013
  W BBa_K4047038 Plasmid pGEM-tal+fbp An Nguyen 10227
  W BBa_K4047039 Plasmid pGEM-SBPase (plasmid for deletion of SBPase) An Nguyen 4616

Wiki Development

A website IDE open with the Miami University's home page code on it.A website editor open with the Miami University's home page code on it. The wiki server has been notorious in iGEM for its difficulty to navigate, and many teams struggle to develop a strong wiki by not being familiar with professional website development. Therefore, we sought to create a platform using code and philosophies that could guide other teams for easier and more directed success. Better wikis also make the important content more accessible to readers, therefore improving upon the goal of scientific communication for iGEM.

First, some code from previous years’ teams were edited to provide a streamlined uploading process that essentially takes word documents, transfers them into markdown files, and uploads them to the wiki server. Many bugs have been resolved and unique difficulties often faced by iGEM teams such as uploading complex mathematical notation are now documented in easily accessible code. Thorough documentation of changes and additions can be found in our publicly accessible Github repository for iGEM-2021-Website under MiamiOH-iGEM.

In addition to the natural content of the code, our documentation and intentionality for the Wiki provide a good example by which other teams can follow. First, the code related to the wiki is documented in a way that clearly expresses how to use the code, with helpful diagrams and a sitemap describing the layout of the site. Exact representation of these detailed descriptions and instructions can be found in our publicly accessible Github repository for iGEM-2021-Website under MiamiOH-iGEM. Similarly, our wiki design is intentional in having information be as accessible as possible. For example, a dictionary was incorporated to allow viewers to simply hover over certain terms and be shown a small definition, serving as easy reminders of some of the more technical terms throughout the wiki. Similarly, all pages were checked to be formatted correctly on all mobile and desktop devices. Rather than describe all philosophies here, we encourage you to look at additional intentions and more thorough descriptions of the previously described steps on the "About the Wiki" Page.

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Communication

We wanted to share our project with others and have an open discussion. Learn how we shared with other communities and what we learned!

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Attributions

Every member of the Miami University iGEM contributed in a meaningful and impactful way. See how and what each person attributed to that made our project what it is!