iGEM 2023 — Team ndsu

### June

| S | M | T | W | T | F | S |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | [20](https://static.igem.wiki/teams/4753/wiki/igem-9-20-notebook.pdf) | 21 | [22](https://static.igem.wiki/teams/4753/wiki/igem-6-22-lab-document.pdf) | 23 | 24 |
| 25 | 26 | [27](https://static.igem.wiki/teams/4753/wiki/6-27-lab-document.pdf) | [28](https://static.igem.wiki/teams/4753/wiki/6-28-lab-document.pdf) | [29](https://static.igem.wiki/teams/4753/wiki/6-29-lab-document.pdf) | [30](https://static.igem.wiki/teams/4753/wiki/6-30-lab-document.pdf) |  |

### July

| S | M | T | W | T | F | S |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | [11](https://static.igem.wiki/teams/4753/wiki/7-11-lab-document.pdf) | [12](https://static.igem.wiki/teams/4753/wiki/7-12-lab-document.pdf) | [13](https://static.igem.wiki/teams/4753/wiki/7-13-lab-document.pdf) | [14](https://static.igem.wiki/teams/4753/wiki/7-14-lab-document.pdf) | [15](https://static.igem.wiki/teams/4753/wiki/7-15-lab-document.pdf) |
| 16 | [17](https://static.igem.wiki/teams/4753/wiki/7-17-lab-document.pdf) | 18 | 19 | 20 | 21 | 22 |
| 23 | [24](https://static.igem.wiki/teams/4753/wiki/7-24-lab-document.pdf) | [25](https://static.igem.wiki/teams/4753/wiki/7-25-lab-document.pdf) | 26 | 27 | 28 | 29 |
| 30 | 31 |  |  |  |  |  |

### August

| S | M | T | W | T | F | S |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | [2](https://static.igem.wiki/teams/4753/wiki/8-2-lab-document.pdf) | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | [14](https://static.igem.wiki/teams/4753/wiki/8-14-lab-document.pdf) | [15](https://static.igem.wiki/teams/4753/wiki/8-15-lab-document.pdf) | 16 | 17 | 18 | 19 |
| 20 | [21](https://static.igem.wiki/teams/4753/wiki/8-21-lab-document.pdf) | 22 | 23 | 24 | [25](https://static.igem.wiki/teams/4753/wiki/8-25-lab-document.pdf) | 26 |
| 27 | [28](https://static.igem.wiki/teams/4753/wiki/8-28-lab-document.pdf) | 29 | 30 | [31](https://static.igem.wiki/teams/4753/wiki/8-31-lab-document.pdf) |  |  |

### September

| S | M | T | W | T | F | S |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |

{% endblock %}

{% extends "layout2.html" %}
{% block page\_content %}

#### Overview

Our project had two genetic approaches: the *purR* Purine Sensor and the Guanine-II Riboswitch. We went through one full engineering cycle and started on a second for both approaches.

---

### *purR* Purine Sensor

---

#### Design

The problem with current methods of measuring varroa mite infestation is their qualitative nature.
Our method should be more accurate than current methods like the “sugar shake”.

By creating a biosensor that produces an increasing amount of reporter pigment that correlates to
the infestation level of the beehive, we can reliably determine infestation levels and intervene
accordingly to save the hive.

Pre-existing natural genetic circuits have been studied and published in peer review articles. Some
of these such as the *tetR* gene have been used and studied so extensively to the point that it is a well
established tool used by genetic engineers. By combining documented sequences centered around guanine
sensing and genetic tools like the *tetR* system, we can create a biosensor based on the extra guanine
in a varroa mite infested beehive.

The CIDAR MoClo Parts Kit has a wide array of established modular components designed to create tailor
made expression vectors through Golden Gate cloning. Designed gene blocks need to be compatible with Golden
Gate cloning in the MoClo scheme to fit the parts available in the CIDAR MoClo Parts Kit.

Our bacterial strain should be easily manipulated for many iterative genetic experiments and is low
maintenance. DH5-α *E. coli* will suffice for early experimentation.

Software for building gene blocks and testing their cloning digitally is necessary. We will use the
Geneious software.

#### Build

The construction of our build will revolve around Golden Gate cloning in the MoClo scheme.

* Fast and efficient method of cloning that relies on type two restriction sites that are excised after ligation which prevents backwards reactions (Golden Gate cloning).
* Genetic segments for all parts can be obtained from online databases and sequences can be edited to contain Golden Gate cloning restriction and recognition sites.

Two parts related to the purR system were identified from literature. The first is a *purR* gene sequence
derived from *E. coli* K12 including a Promoter+RBS+CDS to serve as the guanine sensor and PurR repressor
protein producer [(1)](https://www.jbc.org/article/S0021-9258%2819%2977686-8/pdf). Additionally a
promoter sequence regulated by the PurR repressor protein from the same *E. coli K12* was also identified
[(2)](https://www.sciencedirect.com/science/article/pii/S0021925819776856?ref=pdf_download&fr=RR-2&rr=7bea0a546e51089f)[(3)](https://www.researchgate.net/publication/19854152_Identification_and_nucleotide_sequence_of_a_gene_encoding_5%27-phosphoribosylglycinamide_transformylase_in_Escherichia_coli_K12).

These sequences were then integrated into the MoClo cloning scheme by adding BsaI recognition sites and adding \_AD
overhangs to the guanine sensor sequence and \_AC overhangs to the purR repressor protein regulated sequence.
Additionally, seven extra adenine bases were added to each end of both synthetic sequences to increase cleaving
efficiency [(4)](https://www.neb.com/en-us/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments).

Additionally, a variety of terminators with a range of strengths were chosen from the CIDAR MoClo Extension,
Volume I and modified slightly to have \_DF overhangs instead of \_DE overhangs.

A number of sequences were taken directly and without modification from the CIDAR MoClo Parts kit or CIDAR MoClo
Extension, Volume I. These are parts such as pTet\_AB, BCD2\_BC, c99m (mScarlet-I), and tetR\_CD.

These parts were combined using a BsaI Golden Gate cloning to produce a single expression vector plasmid.
This plasmid was transformed into competent DH5-α *E. coli* cells.

#### Test

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. 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-α is auxotrophic for thiamine.

#### Learn

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.

#### Design

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:
   * Growing our construct in 5ml of M9 media supplemented with 50ul of LB media.
   * Growing our construct in 5ml of M9 media supplemented with 50ul of LB media and 1mM of guanine.
   * 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:
   * Growing our construct in 5.0ml of M9 media supplemented with 1nM of guanine.
   * Growing our construct in 4.995ml of M9 media supplemented with 5ul of LB media and 1nM of guanine.
   * Growing our construct in 4.95ml of M9 media supplemented with 50ul of LB media and 1nM of guanine.
   * Growing our construct in 4.5ml of M9 media supplemented with 500ul of LB media and 1nM of guanine.
   * Growing our construct in 0.5ml of M9 media supplemented with 4.5ml of LB media and 1nM of guanine.
   * Growing our construct in 5.0ml of LB media and 1nM of guanine.
   * 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.
   * 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

---

#### Design

The problem with current methods of measuring varroa mite infestation is the qualitative nature of the
current methods. Our method should be more accurate than current methods like the Sugar Shake.

By creating a biosensor that produces reporter pigment when varroa mite infestation reaches a certain
threshold, we can reliably determine when the infestation level becomes severe enough and intervene to
save the hive before it is too late.

Pre-existing natural genetic circuits have been studied and published in peer review articles. Some of
these such as the *tetR* gene have been used and studied so extensively to the point that it is a well
established tool used by genetic engineers. By combining documented sequences centered around guanine
sensing and genetic tools like the *tetR* system, we can create a biosensor based on the extra guanine
in a varroa mite infested beehive.

The CIDAR MoClo Parts Kit has a wide array of established modular components designed to create tailor made
expression vectors through Golden Gate cloning. Designed gene blocks need to be compatible with Golden Gate
cloning in the MoClo scheme to fit the parts available in the CIDAR MoClo Parts Kit.

Our bacterial strain should be easily manipulated for many iterative genetic experiments and is low maintenance.
DH5-α *E. coli* will suffice for early experimentation.

Software for building gene blocks and testing their cloning digitally is necessary. We will use Geneious
software.

#### Build

The construction of our build will revolve around Golden Gate cloning in the MoClo scheme.

* Fast and efficient method of cloning that relies on type two restriction sites that are excised after
  ligation which prevents backwards reactions (Golden Gate cloning).
* Genetic segments for all parts can be obtained from online databases and sequences can be edited to
  contain Golden Gate cloning restriction and recognition sites.

A Guanine-II Riboswitch gene sequence was derived from *Paenibacillus Sp. HW567*
[(5)](https://www.pnas.org/doi/10.1073/pnas.2120246119). Searching through the genome of the
*Paenibacillus Sp. HW567*, we were not able to find a 100% match to the gene sequences provided in the publication
and the only sequence we could find was incomplete. In an attempt to preserve the sequence cited in the paper,
we used the exact sequences from the paper, then used code from another *Paenibacillus Sp. (FSL R7-0331)* with a
homologous riboswitch region to fill in the gaps left unstated in the paper. The sequence starts with the upstream
86bp from the paper, then a 79bp intergenic region, then the 25bp downstream sequence from the paper, and finally
another 28bp of sequence from *FSL R7-0331* between its downstream region and the coding sequence it modifies.

This sequence was then integrated into the MoClo cloning scheme by adding BsaI recognition sites and \_BC overhangs
to clone it into the RBS slot of a typical Golden Gate design. Additionally, seven extra adenine bases were added
to both ends of the synthetic sequence to increase cleaving efficiency [(6)](https://www.neb.com/en-us/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments).

A number of sequences were taken directly and without modification from the CIDAR MoClo Parts kit or CIDAR MoClo
Extension, Volume I. These are parts such as J23100\_AB and c99m (mScarlet-I).

Additionally, a variety of terminators with a range of strengths were chosen from the CIDAR MoClo Extension,
Volume I and modified slightly to have \_DF overhangs instead of \_DE overhangs.

These parts were combined using a BsaI Golden Gate Cloning to produce a single expression vector plasmid. This
plasmid was transformed into competent DH5-α *E. coli* cells.

#### Test

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 three 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-α is auxotrophic for thiamine.

#### Learn

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.

#### Design

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 maybe there is 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.

---

[### Click for Parts List](https://static.igem.wiki/teams/4753/wiki/partsregistryforwiki-xlsx-sheet1-1.pdf)

---

---

### Best New Composite Part

---

Our team is nominating ourselves for the Best New Composite Part. The part we are nominating is BBa\_K4753012. Ideally we would like to nominate BBa\_K4753011, BBa\_K4753012, and BBa\_K4753013 as they are a trio of pathways that are intended to work together, but just BBa\_K4753012 is one we chooses to nominate.

This composite part promotes the TetR repressor protein based on the amount of PurR repressor in the system. It works as a genetic expression flip to turn the down regulation from the *purR* circuit into increased expression of our reporter *mScarlet*.

BBa\_K4753012 is a composite part made up of the basic parts BBa\_K4753003, BBa\_C0040, and BBa\_K4753005. BBa\_K4753003 is PurMN\_AC, which is a PurR regulated promoter and RBS. Depending on the concentration of environmental PurR, PurMN\_AC changes the expression of a downstream coding sequence. High guanine concentration results in high expression. Low guanine concentration results in low expression. In this scenario, the downstream coding sequence is BBa\_C0040, a region encoding the TetR repressor protein. The final basic part is BBa\_K4753005, A 313x-strength natural terminator. This terminator was only minorly adapted from an existing part from the CIDAR MoClo Extension Vol I.

{% endblock %}

{% extends "layout2.html" %}
{% block page\_content %}

### Competent E. coli Transformation

##### Protocol

1. Thaw a tube of DH5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 2µl containing 1 pg-100 ng of Golden Gate reaction or plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Label and warm selection plates to 37°C.
5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature SOC into the mixture.
8. Shake at 37°C for 1-2 hours. Shake vigorously (250 rpm).
9. Mix the cells thoroughly by flicking the tube and inverting, then perform two 10-fold serial dilutions in SOC.
10. Spread 100µl of each dilution onto a selection plate and incubate overnight at 37°C.

### Gel Electrophoresis

##### Protocol

1. Larger: 120ml 0.5x TAE buffer + 1.8g agarose, Smaller: 60mL 0.5x TAE buffer + 0.9g agarose. Microwave 40-80s, then 5 µL Midori Green
2. Cast the gel in the gel rig if there is the rubber seal, or tape it if you can't find a rubber sealed one. Put in the comb.
3. Ladder = 15µl “1Kb+ GeneRuler” Ladder + 3µl “Purple Loading Dye” dye.
4. Samples (16x) = 25µl of Digest reaction + 5µl “Purple Loading Dye” dye. You can mix dye into the same tube we did the digest in.
5. Once the gel is cooled, take out the comb and orient the gel so that the wells are on the black side. The DNA will “Run to Red”. Fill the gel rig with more 0.5x TAE until it fills and covers the wells.
6. Add ladder + samples to wells.
7. Run at 100V for 60 minutes.
8. Take gel out and image with “UV365”.

### Golden Gate Cloning

##### Protocol

1. Run the Golden Gate mixture in a thermocycler with the program listed in steps 2-4:

   | Reagent | Assembly Reaction (ng/Ul) |
   | --- | --- |
   | DNA parts: Synthesized gene fragments; Plasmids designed for BsaI Golden Gate | 40 fmol |
   | T4 DNA Ligase Buffer (10X) | 2 µl |
   | NEB Golden Gate Enzyme Mix (BsaI-HFv2) | 1.5 µl |
   | Nuclease-free H2O | 20 µl |
2. 30 cycles of: 37°C for 1 minute and 16°C for 1 minute
3. 60°C for 5 minutes
4. Hold at 4°C

### Making Liquid Media

##### Protocol

1. Add LB Lennox Broth powder to MiliQ water in a 20g powder / 1L water ratio. Only make as much as needed.
2. Autoclave the mixture at Liquid20 setting.
3. Once the media has cooled considerably (able to hold bottle comfortably), antibiotics can be added.
4. The concentration of Amp (Ampicillin) in the media is always 100ug/mL unless noted otherwise.
5. The concentration of Spc (spectinomycin) in the media is always 50ug/mL unless noted otherwise.

### Making M9 Mineral Media

##### Protocol

1. Make M9 salt solution by dissolving the appropriate amount of M9 salt into water. The ratio is listed on the bottle the M9 salts come in. Then autoclave the solution
2. Make 20% glucose by dissolving 200g of glucose for each liter of water. Filter sterilize this mixture rather than autoclaving.
3. Make a 1M stock solution for both MgSO4 and CaCl2. These two stocks can be autoclaved.
4. For the biotin and thiamin, the ratio is 1mg/ml. To make a 50ml stock this would be 50mg. For the biotin small amounts of NaOH can be added to properly dissolve it. Both stocks should be filter sterilized rather than autoclaved, and stored at -20C
5. Dissolve 5g of EDTA in 800ml of water. Next add 498mg of FeCl3, and 84mg of ZnCl2
6. This next step requires stocks of CuCl2, CoCl2, H3BO3, and MnCl2 to have been premade, or can be added in mg amounts. MnCl2 is in very small milligram amounts so using stock solution would most likely be easier. Add 765uL of 0.1M CuCl2-2H2O, 210uL of 0.2M CoCl2-6H2O, 1.6mL of 0.1M H3BO3, and 8.1uL of 1M MnCl-4H2O.
7. Solution is made, must be filter sterilized.
8. For 5x M9 Salt solution add 200ml of the M9 salt solution.
9. Next add 20ml of the glucose.
10. Add 2ml of the 1M MgSO4 and 0.1ml of the 1M CaCl2.
11. Add 1ml of the thiamine.
12. Add 1ml of the biotin.
13. Add 10ml of the trace element solution.
14. Fill up the bottle to 1L using sterile water.
15. This is the amount of reagent added to solution to create the specified final concentration

    | Volume | Reagent | Final Concentration |
    | --- | --- | --- |
    | 200 mL | M9 Salt solution (5X) | 1X |
    | 20 mL | 20% glucose | 0.4% |
    | 2 mL | 1M MgSO4 | 2 mM |
    | 0.1 mL | 1M CaCl2 | 0.1 mM |
    | 1 mL | Biotin (1mg/ml) | 1 ug |
    | 1 mL | Thiamin (1mg/ml) | 1 ug |
    | 10 mL | Trace elements solution (100X) | 1X |

### Making Solid Media

##### Protocol

1. Add LB Lennox Broth powder to MiliQ water in a 20g powder / 1L water ratio. Only make as much as needed.
2. Add agar to the the LB and water mix in a 15g agar / 1 L water ratio.
3. Autoclave the mixture at Liquid20 setting.
4. Once the media has cooled considerably (able to hold bottle comfortably), antibiotics can be added.
5. The concentration of Amp (Ampicillin) in the media is always 100ug/mL unless noted otherwise.
6. The concentration of Spc (spectinomycin) in the media is always 50ug/mL unless noted otherwise.

### Plasmid Prep

##### Protocol

1. Pellet 4.5mL culture in a 1.5mL Eppendorf tube by spinning down 1.5mL at a time at 13000rpm and removing supernatant. Repeat 3-4 times. Or spin in a 15mL falcon tube for 10 minutes at 4000g.
2. Resuspend cells in 250uL of buffer P1 (with RNAse added) in an Eppendorf tube
3. Add 250uL Buffer P2 (if it is precipitate, warm the buffer in 30°C incubator) and mix by gently inverting 5 times, incubate at room temperature for exactly 5 minutes
4. Add 350uL Buffer N3 and mix by inverting 5-10 times. Do not vortex. A white precipitant should begin to form.
5. Spin for 10-15 minutes at 13000rpm in a centrifuge until good separation of precipitant and supernatant is achieved.
6. Apply 800uL of clear supernatant to a QIAgen spin column. Avoid the white precipitate.
7. Centrifuge for 60s, discard the flow through.
8. Wash by adding 750uL Buffer PE (with ethanol added) and spinning for 60s. Remove flowthrough.
9. Spin for an additional 2 minutes to remove residual PE buffer and discard the flowthrough.
10. Transfer the top half of the spin column to a new Eppendorf tube. Add 50uL of ddH2O to center of the column, let sit for 3 minutes, then centrifuge for 60s. Discard the spin column.
11. Label the Tube well with at least the following: Sample Name, Date, and Initials;

### Plate Dilutions

##### Protocol

1. 10^-1 plate: 100 µL of original
2. 10^-2 plate: 10 µL of original + 90 µL of S.O.C.
3. 10^0 plate: Spin down remainder @ 13,000 rpm for 1 min, pour off supernatant, resuspend pellet in 100 µL of S.O.C., then plate all 100 µL

### Restriction Digest

##### Protocol

1. 2.5ul rCutSmart Buffer, 0.5µl EcoR, 0.5µl Cla1, 21.5µl DNA.
2. Thermocycler: 37C for 40min then 65C for 20 min.
3. 5 µL rCutSmart Buffer, 5µL Bsa1, 1 µg DNA (1000/concentration), water to 50 µL
4. Thermocycler: 37°C for 5–15 minutes, 80°C for 40 min

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# 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

* Transformation isolates A+B were inoculated into M9
* Isolate B was additionally inoculated into M9+1nM guanine
* Used the white M9 liquid cultures to inoculate LB broth

![results](https://static.igem.wiki/teams/4753/wiki/results-1.png)

![results](https://static.igem.wiki/teams/4753/wiki/results-2.png)

### 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

* Transformation isolates A and B were inoculated into LB
* Transformation isolates A and B were inoculated into M9
* Isolate B was additionally inoculated into M9+1nM guanine

![results](https://static.igem.wiki/teams/4753/wiki/results-3.png)

![results](https://static.igem.wiki/teams/4753/wiki/results-4.png)

### 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

{% endblock %}