Team:Lambert GA/Results

• TEAM
  • TEAM MEMBERS
  • ATTRIBUTIONS
• PROJECT
  • DESCRIPTION
  • DESIGN
  • CONTRIBUTION
  • PROPOSED IMPLEMENTATION
  • ENGINEERING SUCCESS
  • PROOF OF CONCEPT
  • EXCELLENCE IN ANOTHER AREA
  • SUSTAINABLE DEVELOPMENT
  • PARTNERSHIP
• WETLAB
  • PARTS LIBRARY
  • PARTS DESCRIPTION
  • PART COLLECTION
  • PHO
  • NAR
  • PPB
  • EXPERIMENTS
  • NOTEBOOK
  • RESULTS
• DRYLAB
  • HARDWARE
  • MEASUREMENT
  • MODEL
• HUMAN PRACTICES
  • INTEGRATED HUMAN PRACTICES
  • EDUCATION
  • SCIENCE COMMUNICATION
  • COLLABORATIONS
  • SAFETY
• JUDGING FORM
• POSTER

RESULTS

PHO

Characterization Curve

Phosphate concentrations between 50μM and 100μM are ideal for plant growth in hydroponics systems. Our team characterized BBa_K2447000, a phosphate sensor that utilizes the Pho Regulon signaling pathway, through detailed characterization of GFP expression in response to the extracellular phosphate level range of 0μM to 100μM. As shown in Figure 1, The characterization curve of our biosensor exhibited a strong linear negative trend throughout all phosphate concentrations, which closely paralleled the predictive Ordinary Differential Equations model created by our 2020 team.

Figure 1. Characterization curve for BBa_K2447000 for phosphate concentrations between 0-100μM.

Sample Testing

Our phosphate sensor and its characterization curve indicated that the water samples had the following phosphate concentrations:

1. Dick Creek: between 0-10μM
2. Chattahoochee Pointe Park Lake: between 0-10μM
3. Sweetwater Aeroponics System: between 60-70μM

To compare our data to that of a commercial kit, we also utilized the Lamotte phosphate testing kit (Figure 2) and identified each samples’ phosphate concentrations. The results were as follows:

1. Dick Creek: 1μM
2. Chattahoochee Pointe Park Lake: 0μM
3. Sweetwater Aeroponics System: approximately 50μM

The similar results between analysis with our phosphate sensor and a commercial test kit further substantiated that our biosensor can accurately detect extracellular inorganic phosphate levels, validating its usefulness in regulating hydroponics systems.

Figure 2. Phosphate concentration analysis using our water samples and the Lamotte testing kit.

Lyophilization

Using LyphoX, we freeze-dried our phosphate biosensor in order to determine that our cells still function properly after lyophilization. As shown in Figure 3, the biosensor was expressing GFP both before and after lyophilization, demonstrating that LyphoX did not compromise the samples’ cell structure. Additionally, successful sequencing results (Figure 4) showed that the freeze-drying process did not introduce contamination or damage the DNA (See: Proof of Concept).

Figure 3. Visible GFP expression of our phosphate sensor before (left) and after (right) lyophilization

Figure 4. Sequencing results of our phosphate sensor aligned with the original sequence.

NAR/Cell-free

After achieving a plain BL21 cell-free lysate in our high school lab, we tested it with T7 GFP and T7 GFP induced with IPTG. Our lysate was able to produce high fluorescence of T7 GFP without the presence of IPTG and had increased expression with the addition of IPTG, demonstrating that our lysates do work (Figure 5).

Figure 5. Plate Reader fluorescence data for T7 GFP and T7 GFP + IPTG

After the development of the cell-free lysate of the plain BL21 cells, we prepared a NarX enriched lysate to implement our nitrate sensor into our cell-free system. (Figure 6). We have been testing our nitrate sensor with nitrate concentrations from 0 ppm to 300 ppm and are still in the process of experimentation.

Figure 6. NarX Enriched Cell-Free Lysates

In addition, with the success of our BL21 cell lysate, we have begun initial experimentation to implement our Fusarium and Phytophthora plant pathogen biosensors into a cell-free system.

PPB

Improved Fusarium Toehold with GFP Reporter

We performed electrocompetent dual plasmid transformation on part BBa_K3725022, Improved Fusarium Toehold w/ GFP Reporter, and part BBa_K3725070, T7 Fusarium Trigger using kanamycin-carbenicillin plates to select for cells that had taken up both plasmids.

Figure 7. Fusarium Pair 2 Dual Plasmid transformation.

We made liquid cultures of the Fusarium pair 2 dual plasmid cells, performed a miniprep, and a restriction digest(Figure 7). The gel electrophoresis confirmed the success of our dual plasmid transformation(Figure 8).

Figure 8. Gel confirming successful dual plasmid transformation of Improved Fusarium toehold switch and trigger. Well 5 is the standardized DNA ladder, and well 6 contains the Improved Fusarium toehold w/ GFP reporter and trigger at approximately 2,800 bp and 2,000bp, respectively.

To test whether our dual plasmid transformation was successful, we measured and compared the fluorescence and optical density (OD) of the dual plasmid cells, toehold, and pUC19 cells. In order for the dual plasmid transformation to be deemed successful, the fluorescence/OD of the dual plasmid cells should be significantly different compared to that of the toehold, trigger, and pUC19 cells. The obtained measurements of fluorescence/OD for the dual plasmid cells were greatly significantly different (according to SEM bars) from the measurements of the cells transformed with only the toehold and cells transformed with the positive control, pUC19 (Figure 9).

Figure 9. Mean fluorescence/OD of IPTG-induced Fusarium pair 2 dual plasmid transformation compared to toehold and pUC19 with SEM error bars. Ran at gain of 40.

Dual Plasmid Transformation- Phytophthora Toehold with GFP Reporter

We performed electrocompetent dual plasmid transformation (Figure 10) on part BBa_K3725010, Phytophthora Toehold w/ GFP Reporter, and part BBa_K3725040, T7 Phytophthora Trigger using kanamycin-carbenicillin plates to select for cells that had taken up both plasmids.

Figure 10. Transformation of Phytophthora Toehold w/ GFP reporter (left) compared to Phytophthora Dual Plasmid transformation (right).

We grew liquid cultures of the dual plasmid cells and performed a miniprep that gave optimal concentration and purity values. By performing a restriction digest on the miniprep product, we demonstrated that both vectors were successfully cloned into the cell (Figure 11).

Figure 11. Gel confirming successful dual plasmid transformation of Phytophthora toehold switch and trigger. Wells 3 and 6 contain NEB 1kb standardized DNA ladders, and wells 1, 2, 4, and 5 contain the Phytophthora toehold and trigger at 2,800bp and 2,000bp, respectively.

To test whether our dual plasmid transformation was successful, we measured and compared the fluorescence and optical density (OD) of the dual plasmid cells, toehold, and pUC19 cells. In order for the dual plasmid transformation to be deemed successful, the fluorescence/OD of the dual plasmid cells should be significantly different compared to that of the toehold, trigger, and pUC19 cells. The obtained measurements of fluorescence/OD for the dual plasmid cells were greatly significantly different (according to SEM bars) from the measurements of the cells transformed with only the toehold and cells transformed with the positive control, pUC19 (Figure 12).

Figure 12. Mean fluorescence/OD of IPTG-induced Phytophthora dual plasmid transformation compared to toehold and pUC19 with SEM error bars. Ran at gain of 60.