Results

purR Purine Sensor

Introduction

In order to determine if our purR final construct would successfully produce mScarlet protein in response to guanine concentration, we transformed the construct into competent E. coli cells and inoculated different types of media with 3 isolates of the purR final construct.

Experiments

results
results

Analysis

Our purR construct produces the mScarlet reporter protein in LB media but not in M9 media or M9 media supplemented with guanine. This could mean a few things:

  1. The guanine we are using is not the correct form of guanine needed to trigger the purR guanine sensor and the correct form is found within the more complex LB media.
  2. The purR-tetR-mScarlet pathway needs at least trace amounts of some nutrient found in the LB media and absent in the M9 media in order to fully carry out the pathway from sensing the guanine to expressing the mScarlet reporter.
  3. The purR pathway could be sensing and reacting to the concentration of a substrate other than guanine that is found in LB media and not the M9 media.

Future Directions

Before completely re-designing the purR construct, we need to carry out further testing to determine what is wrong with the first iteration.

  1. To address the first potential issue, we could order different forms of guanine and repeat testing with a different supplemental guanine.
  2. To address the second potential issue, we could try:
    1. Growing our construct in 5ml of M9 media supplemented with 50ul of LB media.
    2. Growing our construct in 5ml of M9 media supplemented with 50ul of LB media and 1mM of guanine.
    3. If a clear difference in the saturation of red color in the broth is observed, then the issue is likely that M9 is too minimal of a media to support at least one part of the purR-tetR-mScarlet pathway.
  3. To address the third potential issue, we could try a similar experiment to issue 2:
    1. Growing our construct in 5.0ml of M9 media supplemented with 1nM of guanine.
    2. Growing our construct in 4.995ml of M9 media supplemented with 5ul of LB media and 1nM of guanine.
    3. Growing our construct in 4.95ml of M9 media supplemented with 50ul of LB media and 1nM of guanine.
    4. Growing our construct in 4.5ml of M9 media supplemented with 500ul of LB media and 1nM of guanine.
    5. Growing our construct in 0.5ml of M9 media supplemented with 4.5ml of LB media and 1nM of guanine.
    6. Growing our construct in 5.0ml of LB media and 1nM of guanine.
    7. If the sensor is sensing something other than guanine, then we would expect to see that the red color of the broth increases in parallel with the increasing concentration of the LB broth.
    8. If the sensor is truly sensing guanine, then we would expect to see a large increase in the red color of the broth as soon as it reaches the amount LB needed to activate the part/s of the purR-tetR-mScarlet pathway.

Guanine-II Riboswitch


Introduction

In order to determine if our Guanine-II Riboswitch final construct would successfully produce mScarlet protein in response to guanine concentration, we transformed the construct into competent E. coli and inoculated different types of media with 3 isolates of the purR final construct. Each isolate was inoculated into LB, LB+1nM guanine, M9, and M9+1nM guanine. Additionally, all of the M9 media included 1% thiamine as DH5-alpha is auxotrophic for thiamine.

Experiments

results
results

Analysis

The riboswitch seems to be completely or near completely non-functional. The constitutive promoter is producing the mScarlet protein regardless of the media type or guanine concentration in the media.

Future Directions

Re-design the SynRiboswitch_BC part. Instead of trying to conserve the sequence listed in the publication, just use the natural and uninterrupted sequence in the genome of the Paenibacillus Sp. HW567. It may not be exactly as listed in the publication, but there may be an error in the publication. The actual sequence taken from the genome should be functional. They studied that strain after all. In other words, align the sequence from the publication to the Paenibacillus Sp. HW567 genome and find the closest match. Take that sequence up until the first stop codon encountered after the aligned region and add BsaI Golden Gate sites to give it _BC overhangs. Another approach would involve looking further into the literature to find any other examples of guanine sensing riboswitches and attempting the same with them.


Supplementary Experiments

Introduction

The two sections above are the primary components we tested. In addition to this, we conducted supplementary experiments and have plans for other future experiments that would improve our understanding of the hive environment. These experiments include quantifying guanine in the hive to find background guanine content as well as understand how much varroa mite infestation will increase guanine concentration by. This will allow us to fine tune our testing process to create pigment at specific guanine concentrations, rather than giving false positives due to background guanine.

Experiments

Accompany USDA field manager as they counted varroa mite counts using non-lethal powdered sugar test at the beginning and near the end of the season.

Hive # # Mites (June 27) # Mites (August 2)
3 0 1
6 0 1
11 0 1
12 0 5

One cup is approximately 100 bees, and mite levels should be kept below 1 mite/100 in spring and 2 mites/100 bees for the rest of the year. (University of Minnesota)

Analysis:

The results from sampling the bees at two different time points shows how the amount of varroa mites increased from 0 mites early in the year (6/27) to an average of 2 mites for every 100 bees later in the year (8/2). This testing method may not be completely accurate, due to the proximity of these hives to each other and the tendency for hives close to each other to have similar levels of varroa mite infection (M.A. Stevenson, et al). The potential inaccuracy of this test is part of why there is a need for a new testing method to be developed.

Future Directions:

Now that an infected population has been established, next step experiments would involve using these hives to determine guanine concentration in the hive based on varroa mite infestation level. This would involve continuing to monitor the varroa mite infection within the hive. Brood cells would then be swabbed to as samples for high performance liquid chromatography to determine guanine levels in the hive. Once guanine concentration was found for specific levels of infection we could fine tune the production of mScarlet to only be created at levels of guanine indicating a varroa mite infection. This would prevent false positives by calibrating our design to only start production under correct conditions.

Conclusion:

We conducted experiments to understand the guanine sensing capabilities of our new part, and supplementary experiments to understand the parameters of our environmental conditions. The majority of our experiments were unsuccessful, but they gave us useful insight on how we can improve our design. Using these results, we know what changes to make to our designs and experimental processes. On top of this, there are multiple other experiments that would be conducted to fine tune and finalize our designs.

Works Cited

  1. M.A. Stevenson, H. Benard, P. Bolger, R.S. Morris, Spatial epidemiology of the Asian honey bee mite (Varroa destructor) in the North Island of New Zealand, Preventive Veterinary Medicine, Volume 71, Issues 3–4, 2005, Pages 241-252, ISSN 0167-5877
  2. University of Minnesota, UM, Varroa Mite Testing & Management. Varroa Mite Testing & Management | Bee Lab. (2023). https://beelab.umn.edu/varroa-mite-testing