Experiments



  This season, our team conducted experiments for 1) improvement of an existing part and 2) for our proof of concept.


1) Improvement of an Existing Part


Original Part:

    Original osmY Stationary Phase Detection Construct from MIT iGEM 2006

      The original composite part, which was designed by MIT iGEM 2006, can be found on the iGEM Registry as part BBa_J45995. This construct consists of the following basic parts: an osmY promoter, ribosome binding sequence BBa_B0030, a green fluorescence protein (GFP) coding region, and terminator BBa_B0015. Between the promoter and RBS, RBS and coding region, and coding region and terminator, the DNA sequence contains scar sequences from assembly.

      The promoter selected was an osmY promoter, as this promoter is induced by the cell’s entry into stationary phase. In typical E. coli cells, osmY, which helps cells transition into stationary phase when under osmotic or metabolic stress, is not produced during exponential growth phase but is produced during stationary phase. Specifically, the osmY promoter is induced by rpoS (ribosome polymerase sigma S) at the onset of stationary phase (Chang 2002). Because the coding region in this construct is GFP, this construct fluoresces green once the cell enters stationary phase.



Design:


    A. osmY-sfGFP Stationary Phase Detection Construct (BBa_K4174002)

      We improved this part by [a] increasing fluorescence by replacing GFP with superfolder GFP (sfGFP), [b] removing non-functional scar sequences, and [c] making the sequence compatible with a new assembly method (Type IIS).

      a. Replacing GFP with sfGFP and Changing the RBS

        Superfolder GFP (sfGFP) is an improved version of GFP which folds more readily and precisely in Escherichia coli, allowing for more efficient, bright, and accurate assays (Pédelacq 2006). We sourced our sfGFP sequence from Ceroni et al. (2015). This sfGFP sequence was designed for high-level expression in E. coli (Ceroni et al. 2015). In order to ensure that our ribosome binding sequence (RBS) was compatible with our coding region, we also replaced the original RBS with an RBS used by Ceroni et al. (2015) with their sfGFP sequence.

      b. Removing Non-functional Scar Sequences

        The original construct sequence has scar sequences present from assembly. These nonfunctional units were removed by our team as they are purely artifacts of biological assembly.

      c. Making the Construct Type IIS Assembly Compatible

        All constructs uploaded to the iGEM Registry that are considered for the iGEM competition must be compatible with either Type IIS assembly or RFC 10 Assembly. The original MIT iGEM 2006 construct (BBa_J45995) was only compatible with RFC 10, but after altering the sequence as described above to create our composite part, it is now compatible with both Type IIS Assembly and RFC 10 Assembly. Increasing the options for assembly methods compatible with our construct will help make its construction more accessible to researchers.


    B. osmY-mRFP1 Stationary Phase Detection Construct (BBa_K4174001)

      We improved this part by [a] offering a new fluorescence option to researchers by replacing GFP with monomeric red fluorescence protein (mRFP1) and [b] making the sequence compatible with a new assembly method (Type IIS).

      a. Replacing GFP with mRFP1

        Creating alternative versions of fluorescent bioreporters using proteins with different excitation and emission wavelengths allows researchers to assay multiple parameters at a time, as different fluorescent assays conducted simultaneously must use proteins with different absorption spectra in order for researchers to differentiate between them. We replaced GFP with mRFP1 in order to give researchers a red fluorescence protein for detecting stationary phase in bacteria. mRFP1 is a monomer, has a rapid maturity rate, and has minimal spectral overlap with GFP compared to the wild-type red fluorescent protein DsRed (Campbell et al. 2002).

      b. Making the Construct Type IIS Assembly Compatible

        All constructs uploaded to the iGEM Registry that are considered for the iGEM competition must be compatible with either Type IIS Assembly or RFC 10 Assembly. The original MIT iGEM 2006 construct (BBa_J45995) was only compatible with RFC 10 Assembly, but after altering the sequence as described above to create our composite part, it became compatible with both Type IIS Assembly and RFC 10 Assembly. Increasing the options for assembly methods compatible with our construct will help make its construction more accessible to researchers.



Build:

  In order to assemble the original MIT iGEM 2006 construct (BBa_J45995) along with our improved constructs, we selected to use Gibson Assembly. Our inserts (described in the design section above) were synthesized by IDT and were flanked by unique nucleotide sequences (UNSs) 1 and 10 (Torella et al. 2014). Our backbone, pSB1C3, was also flanked by UNSs 1 and 10. We isolated this backbone from a 2018 William and Mary iGEM construct using PCR primers that anneal to the UNS sequences, then performed a gel extraction and PCR purification before utilizing the backbone for Gibson Assembly. Please download our protocols for PCR, gel extraction, PCR purification, transformation of E. coli and Gibson Assembly below.

Download Protocols Below:



Test:

    Protocol for Testing Parts

    To test the effectiveness of our improved parts, our team grew all three constructs in E. coli NEB5-α cells in our plate reader. They were grown at 37°C, and were continuously shaking in a plate reader (with OD600 and fluorescence measurements taken every 10 minutes). Although the cultures were grown for around a total of 19 hours and 35 minutes, the graphs shown below only include measurements taken starting from around the 10 hour and 5 minute mark. In addition, the graphs below represent the averages of fluorescence measurements (normalized to OD600) taken from two experiments. However, the inoculation process differed between these two experiments. For one of our experiments, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of LB, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For both experiments, 200 uLs of culture was loaded into each well. Also, for both experiments, green fluorescence was measured using an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and red fluorescence was measured using an excitation wavelength of 584 nm and an emission wavelength of 610 nm. Please download our raw fluorescence data and protocols for preparation of LB media, inoculation, and plate reader testing below.

    Download Raw Fluorescence Data Below:

    Download Protocols Below:



    Results:


      BBa_K417002 Characterization (osmY-sfGFP Construct)

        To test the effectiveness of our osmY-sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 construct, our improved osmY-sfGFP construct, and our improved osmY-mRFP1 construct (BBa_K4174001) in E. coli NEB5-α in a plate reader. The various transformants were grown at 37°C with continuous shaking. For green fluorescence, we used an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The values for green fluorescence intensity normalized to OD600 values are reported below.

      The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) taken from two experiments. Please note that the inoculation process differed between these two experiments (see the protocol section of this page for details).

        As seen in the graph above, both the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs enter stationary phase right before 14 hours, but our improved osmY-sfGFP construct (BBa_K4174002) is much more fluorescent. The other constructs are our osmY-RFP construct (BBa_K4174001) and untransformed E. coli cells, both of which serve as negative controls for green fluorescence.

      As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct (BBa_J45995). Here, bacterial cells transformed with our osmY-sfGFP (BBa_K4174002) construct are on the far right, and are visibly more green than cells transformed with the original MIT iGEM 2006 construct (BBa_J45995).


      BBa_K417001 Characterization (osmY-mRFP1 Construct)

        To test the effectiveness of our osmY-mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 construct (BBa_J45995), our osmY-mRFP1 construct, and our osmY-sfGFP construct (BBa_K4174001) into E. coli NEB5-α cells and grew the various transformants in a plate reader. The various transformants were grown at 37°C with continuous shaking. For red fluorescence measurements, we used an excitation wavelength of 584 nm and an emission wavelength of 610 nm. The values for red fluorescence intensity normalized to OD600 values are reported below.

      The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) taken from two experiments. Please note that the inoculation process differed between these two experiments (see the protocol section of this page for details).

      Based on the graph above, the bacterial cells engineered with our osmY-mRFP1 construct (BBa_K4174001) appear to have entered stationary phase around 16 hours. As seen in the graph, our osmY-mRFP1 construct (BBa_K4174001) produces more red fluorescence than the original construct (BBa_J45995) (as expected since the original construct was a GFP construct). The other measurements taken are for our osmY-sfGFP construct (BBa_K4174002) and untransformed E. coli NEB5-α cells, both of which serve as negative controls for red fluorescence.

      The fluorescence intensity of the untransformed cells appears higher than that of cells transformed with our osmY-mRFP1 construct due to our normalization process. Although the raw fluorescence values of the untransformed cells were consistently lower than the raw fluorescence values of the osmY-mRFP1 circuit, the OD600 values of the untransformed cells were much lower than the OD600 values of the cells transformed with osmY-mRFP1. Therefore, when we normalized by dividing raw fluorescence by OD600, the fluorescence intensity of the untransformed cells appeared to be higher than that of the osmY-mRFP1 transformants.



      Based on the graph above, the bacterial cells engineered with our osmY-mRFP1 (BBa_K4174001) construct appear to have entered stationary phase around 16 hours. As seen in the graph, our osmY-mRFP1 (BBa_K4174001) construct produces more red fluorescence than the original construct (BBa_J45995) (as expected since the original construct was a GFP construct). The other measurements taken are for our osmY-sfGFP construct (BBa_K4174002) and untransformed E. coli NEB5-α cells, both of which serve as negative controls for red fluorescence.


      As seen in the image above, qualitative results reveal that our osmY-mRFP1 construct (BBa_K4174001) produces visibly red flourescence. Here, bacterial cells transformed with our osmY-mRFP1 construct are on the far left alongside the other two cultures transformed with the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs.


2) Experiments for 16S rRNA Sequencing


Overview

  The majority of the data used to train and validate our software was obtained from online databases and individual studies. As we were searching for our data, we noticed that much of this sequencing data lacks corresponding environmental parameters (ex. soil temperature, moisture-content, and carbon-content). For our team’s proof of concept, we went to the field and collected 12 soil samples along with their associated parameters. After DNA soil extraction, the soil samples were sent to Dr. Randolph Chambers to have specific parameters analyzed and recorded. 16S PCR was performed on these soil extractions. Primer pair (f 5’- CCTACGGGNGGCWGCAG –3’) and (r 5’- GACTACHVGGGTATCTAATCC -3’) were used targeting the 16s rRNA V3-V4 region. These PCR products were then sent out for sequencing. Combining the sequencing results and the soil parameters, we tested our software’s functionality. In addition to using this data to test our software, we also plan to make it available to other researchers.

1) Selection of 16S PCR Primers

Picking the optimal primer set is a crucial step when setting up 16S rRNA gene sequencing. For our project, we depended heavily on our review of the literature when deciding which primer set to utilize. Among the 22 papers we read, 16 of them conducted 16S rRNA gene sequencing specifically for the purpose of bacterial diversity determination in soil. The purpose of these papers varies from comparing 16S primer pairs in silico to actually utilizing 16S technology in soil samples. After carefully studying these papers, we believe that our primer set should aim to amplify the V3-V4 amplification region. The V3-V4 hypervariable region has been mentioned by 17 out of 22 papers we read on testing soil samples obtained from various regions using different sequencing platforms. In an evaluation of four 16S primers pairs, one paper concluded that “341f/785r [primers targeting the V3-V4 region] detected the highest bacterial diversity, broadest taxonomic coverage, and provided the most reproducible results” (Thijs et al. 2017). Other researchers have also seemed to reach a consensus that primer pairs amplifying the V3-V4 region are “preferred” as they are accurate and have been used extensively across the field (Wang et al. 2018, Xia et al. 2019). Orwin et al. also stated that due to the popularity of these primer pairs, “the extensive reference databases for the V3-V4 region of the microbial 16S rRNA gene have allowed information about soil bacterial communities to be obtained at a much finer taxonomic resolution” (Orwin et al. 2018). Using a widely used primer pair not only provides us with many protocols from scientific literature to reference for our experimental design, but also aids our data analysis through the existing reference databases for our region of interest. Among the primer pairs we found targeting the V3-V4 region, the primer pair (f 5’- CCTACGGGNGGCWGCAG –3’) and (r 5’- GACTACHVGGGTATCTAATCC -3’) was used by 10 papers in addition to the Illumina 16S metagenomic sequencing library preparation protocol. Due to the accuracy of these primers and their consistent use in the literature, we selected a primer pair amplifying the V3-V4 region of the 16S rRNA gene for our own 16S rRNA sequencing experiments.

Download Sources from our 16S PCR Literature Review Below:

  • 16S Literature Review Sources

    2) Soil Sample Collection

      1. The soil samples were collected along the Matoaka Trails surrounding Lake Matoka, Williamsburg, Virginia. These trails cover a range of soil types including Alluvium, Norfolk Formation, Windsor Formation, Bacon’s Castle Formation, Sedley Formation, Yorktown Formation, and Saint Marys Formation.

      2. At each soil digging site, soil parameters including coordinates, sample collection time, latitude and longitude (GPS), sample depth, elevation, altitude, humidity, soil temperature, air temperature, and pH were collected.

      3. Using a washed shovel, dig and collect the soil into a plastic collection bag. Collect soil horizontally to ensure the entire soil sample came from a uniform depth.

      4. Moisture content was measured using an oven-baked method. Weights of the samples were measured before and after oven-drying.

      5. Portions of the samples were sent to Dr. Randy Chambers for measuring the sample’s organic content.

    Please download our protocol for soil sample collection below:

    • Soil Sample Collection Protocol

      3) Soil DNA Extraction

        1. Sieve soil

        2. Two replicates for each soil sample, one extracted using Qiagen DNAeasy Soil Extraction Kit, one extracted using Qiagen DNeasy PowerSoil Pro Kit

        3. Nanodrop/Gel Confirmation of DNA Extraction

      Please download our protocols for soil DNA extraction below:

    • Soil DNA Extraction Protocol - Qiagen DNAeasy Soil Extraction Kit
    • Soil DNA Extraction Protocol - Qiagen DNeasy PowerSoil Pro Kit

      4) 16S PCR


      1. Two settings were used (see Table 1 below)
        2. Source Initial Denaturation Denaturation Primer Annealing Extension Final Extension
        Settings from Sinclair, Bertilsson, & Eiler 2015 95°C for 5s 20 cycles at 95°C for 40 s 53°C for 40s 72°C for 60s 72 °C for 7 min
        Modified Settings 98°C for 30s 20 cycles at 98°C for 10s 53°C for 40s 72°C for 60s 72 °C for 7 min

        Table 1: Top row contains PCR settings from Sinclair Sinclair, Bertilsson, & Eiler 2015; bottom row contains PCR settings modified from Sinclair, Bertilsson, & Eiler 2015.

      2. PCR Purification with NEB Monarch Kit.
      3. Purified PCR products were sent for sequencing.

        4. Please download our protocol for 16S PCR here: PCR for 16S rRNA Sequencing Protocol



References


Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., & Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 99(12), 7877–7882. https://doi.org/10.1073/pnas.082243699

Ceroni, F., Algar, R., Stan, G., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature Methods, 12(5):415-418. Doi: 10.1038/nmeth.3339

Chang, D. E., Smalley, D. J., & Conway, T. (2002). Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model. Molecular microbiology, 45(2), 289-306.

Illumina. (n.d.). 16S Metagenomic Sequencing Library Preparation. Illumina.com. Retrieved October 9, 2022, from https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

Jaishankar, J., & Srivastava, P. (2017). Molecular basis of stationary phase survival and applications. Frontiers in microbiology, 8, 2000.

Orwin, K.H., Dickie, I.A., Holdaway, R., & Wood, J.R. (2018). A comparison of the ability of PLFA and 16S rRNA gene metabarcoding to resolve soil community change and predict ecosystem functions. Soil Biology and Biochemistry, 117, 27-35. https://doi.org/10.1016/j.soilbio.2017.10.036.

Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology, 24(1), 79-88.

Sinclair, L., Osman, O. A., Bertilsson, S., & Eiler, A. (2015). Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the illumina platform. PloS one, 10(2), e0116955. https://doi.org/10.1371/journal.pone.0116955

Thijs, S., Op De Beeck, M., Beckers, B., Truyens, S., Stevens, V., Van Hamme, J. D., Weyens, N., & Vangronsveld, J. (2017). Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys. Frontiers in microbiology, 8, 494. https://doi.org/10.3389/fmicb.2017.00494

Wang, F., Men, X., Zhang, G., Liang, K., Xin, Y., Wang, J., Li, A., Zhang, H., Liu, H., & Wu, L. (2018). Assessment of 16S rRNA gene primers for studying bacterial community structure and function of aging flue-cured tobaccos. AMB Express, 8(1), 182. https://doi.org/10.1186/s13568-018-0713-1

Xia, X., Zhang, P., He, L., Gao, X., Li, W., Zhou, Y., Li, Z., Li, H., & Yang, L. (2019). Effects of tillage managements and maize straw returning on soil microbiome using 16S rDNA sequencing. Journal of integrative plant biology, 61(6), 765–777. https://doi.org/10.1111/jipb.12802

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