iGEM 2021 — Team Stony_Brook

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# Team:Stony Brook/Notebook

iGEM SBU 2021

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![](https://static.igem.org/mediawiki/2021/4/4f/T--Stony_Brook--pacmanpreloader.gif)

# Notebook

## Weekly Summaries

1. [Weeks 1, 2, 3 summary](https://static.igem.org/mediawiki/2021/1/17/T--Stony_Brook--week1.pdf)- [Week 4 summary](https://static.igem.org/mediawiki/2021/5/54/T--Stony_Brook--week4.pdf)
   - [Week 5 summary](https://static.igem.org/mediawiki/2021/6/68/T--Stony_Brook--week5.pdf)
   - [Week 6 summary](https://static.igem.org/mediawiki/2021/3/34/T--Stony_Brook--week6.pdf)
   - [Week 7 summary](https://static.igem.org/mediawiki/2021/f/f3/T--Stony_Brook--week7.pdf)
   - [Week 8 summary](https://static.igem.org/mediawiki/2021/2/2f/T--Stony_Brook--week8.pdf)
   - [Week 9 summary](https://static.igem.org/mediawiki/2021/8/8b/T--Stony_Brook--week9.pdf)

## Wet Lab notebook

- [Anchoring Construct Notebook](https://static.igem.org/mediawiki/2021/8/85/T--Stony_Brook--anchor.pdf)
- [PP1 Assay notebook](https://static.igem.org/mediawiki/2021/6/66/T--Stony_Brook--PP1assay.pdf)
- [TatExpress Protocol](https://static.igem.org/mediawiki/2021/6/6f/T--Stony_Brook--TATprotocol.pdf)

## Dry Lab Notebook

- [Deepnote notebook](https://static.igem.org/mediawiki/2021/f/f1/T--Stony_Brook--Deepnote_Notebook.pdf)

![](https://static.igem.org/mediawiki/2021/a/a1/T--Stony_Brook--iGEM_v3.jpeg)

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[![](https://static.igem.org/mediawiki/2021/b/ba/T--Stony_Brook--IDT_logo.jpeg)](https://www.idtdna.com/pages)

[![](https://static.igem.org/mediawiki/2021/a/a4/T--St_Andrews--snapgene.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2021/4/40/T--Stony_Brook--Twist.jpeg)](https://www.twistbioscience.com)

[![](https://static.igem.org/mediawiki/2021/f/f9/T--Greece_United--geneious.png)](https://www.geneious.com)

[![](https://static.igem.org/mediawiki/2021/0/0a/T--Stony_Brook--Promega.jpeg)](https://www.promega.com/)

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[![](https://static.igem.org/mediawiki/2021/a/a4/T--St_Andrews--snapgene.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2021/4/40/T--Stony_Brook--Twist.jpeg)](https://www.twistbioscience.com)

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# Team:Stony Brook/Engineering

iGEM SBU 2021

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[Implementation](https://2021.igem.org/Team%3AStony_Brook/Implementation)

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[Parts Overview](https://2021.igem.org/Team%3AStony_Brook/Parts)
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Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3AStony_Brook/Human_Practices)
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[Education & Communication](https://2021.igem.org/Team%3AStony_Brook/Communication)
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[Notebook](https://2021.igem.org/Team%3AStony_Brook/Notebook)
[Model](https://2021.igem.org/Team%3AStony_Brook/Model)
[Results](https://2021.igem.org/Team%3AStony_Brook/Results)
[Engineering Success](https://2021.igem.org/Team%3AStony_Brook/Engineering)
[Implementation](https://2021.igem.org/Team%3AStony_Brook/Implementation)

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[Parts Overview](https://2021.igem.org/Team%3AStony_Brook/Parts)
[Contribution](https://2021.igem.org/Team%3AStony_Brook/Contribution)

[Safety](https://2021.igem.org/Team%3AStony_Brook/Safety)

Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3AStony_Brook/Human_Practices)
[Collaborations](https://2021.igem.org/Team%3AStony_Brook/Collaborations)
[Education & Communication](https://2021.igem.org/Team%3AStony_Brook/Communication)
[Partnership](https://2021.igem.org/Team%3AStony_Brook/Partnership)

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# Engineering Success

Design
Build
Test
Learn

## Anchoring Construct

For the PgsA-MlrA construct, a plasmid was designed to express MlrA on the membrane surface of *E. coli*. A DNA insert containing the anchoring and enzyme genes was attached to a DNA backbone via HiFi assembly to form the final plasmid. Firstly, this insert contains a ptac promoter, which is induced by IPTG (Browning et al., 2017). An inducible promoter was used because a constitutive promoter could run the risk of killing the bacteria by depleting its resources. To ensure protein expression, an Anderson family ribosome binding site (BBa\_J61100) was used because it is suitable for general protein expression in *E. coli*. Next, the DNA insert contains the PgsA (anchoring motif) gene connected to the MlrA (microcystinase) gene with a 2 amino acid Gly-Ser linker. PgsA (BBa\_K2963020), the GS linker (BBa\_J18920), and MlrA (BBa\_K1907002) were parts from the registry used to form a new composite part. To ensure these genes can be expressed in *E. coli*, this composite part was codon optimized via IDT’s codon optimization tool. The terminator.. Overhangs were built into the PgsA-MlrA gBlock to avoid an additional PCR step that could potentially introduce errors in the sequence.

![](https://static.igem.org/mediawiki/2021/3/38/T--Stony_Brook--PgsAinsert.jpeg)

**Figure 1.** Final DNA insert for PgsA-MlrA.

For the backbone or vector pKSI-1 was used because of its efficiency in bacterial expression and high copy number. This vector has ampicillin resistance.

For the *E. coli* chassis, it was decided to use BL21(DE3). BL21(DE3) is commonly used for recombinant protein expression because it contains a T7 RNA polymerase gene under the control of a lacUV5 promoter in the bacterial genome, which allows for high protein expression (Rosano et al., 2019).

![](https://static.igem.org/mediawiki/2021/a/a7/T--Stony_Brook--PgsAPlasmid.jpeg)

**Figure 2.** Final plasmid for the Anchoring Construct.

## TatExpress: Periplasmic Secretion

The design of the TatExpress construct was based on the paper published by Browning et al., “Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway”. As mentioned on the contribution page of the wiki, heterologous expression of MlrA typically results in the protein being localized to the cytoplasm ((Dziga et al., 2012)). Given that microcystin is found extracellularly and does not freely diffuse into the cytoplasm, this results in limited exposure of MC-LR to MlrA and, ultimately, inefficient degradation. In creating the TatExpress strain described by Browning et al., this issue could be addressed as machinery to export MlrA would be upregulated.

The promoter chosen for the insert was pTet (BBa\_K3171173), as it can be used as both an inducible and constitutive promoter. The insert also included a TorA signal sequence and mature four amino acid TorA protein. These sequences were obtained from Browning’s 2017 paper, but a similar part can be found in the registry (BBa\_K1012002). This signal peptide, fused to the N-terminus of MlrA, would allow MlrA to be transported via the Tat machinery. Finally, the RBS and terminator used were BBa\_J61100 and BBa\_B0010, respectively.

![](https://static.igem.org/mediawiki/2021/f/ff/T--Stony_Brook--tatplasmid.jpeg)

**Figure 3.** Final plasmid for the TatExpress construct.

## Detection System

The detection portion of the project was based on the bacterial two-hybrid system and required three inserts: one for each half of the adenylyl cyclase catalytic domain, and one encoding for green fluorescent protein (GFP), which would be expressed in the presence of microcystin.

The first insert codes for the catalytic domain of protein phosphatase 1 (PP1) and one fragment of the adenylyl cyclase catalytic domain, T18. A strong constitutive promoter from the Anderson family was used (BBa\_J23112), followed by an Anderson family RBS (BBa\_J61102), the gene for PP1 (BBa\_K1012001), a 54 amino acid Gly-Gly-Ser linker (BBa\_K3128010), and the gene for T18 (BBa\_K1638004). Finally, an rrnBT1-T7Te terminator (BBa\_B0015) was used.

![](https://static.igem.org/mediawiki/2021/3/38/T--Stony_Brook--Dinsert.jpeg)

**Figure 4.** First insert contains the genes for PP1 and T18.

The second insert codes for glutathione (GSH) and the other fragment of the adenylyl cyclase catalytic domain, T25. The parts used were a constitutive promoter (BBa\_J23112), RBS (BBa\_J61102), GSH (BBa\_K2571001), T25 (BBa\_K1638002), and rnpB-T1 terminator (BBa\_J61048) were used.

![](https://static.igem.org/mediawiki/2021/a/af/T--Stony_Brook--Dinsert2.jpeg)

**Figure 5.** Second Insert contains the genes for GSH and T25.

The last insert codes for green fluorescent protein (GFP) and is responsible for the output of the detection system. A cAMP inducible promoter was used (BBa\_K121011), followed by an RBS (BBa\_J61102), the gene for GFP (BBa\_E0040), and an rrnB T1 terminator (BBa\_B0010).

![](https://static.igem.org/mediawiki/2021/7/76/T--Stony_Brook--Dinsert3.jpeg)

**Figure 6.** Last Insert contains the gene for green flurescent protien (GFP.

Initially during the design process, all three inserts had the same terminator. However, it was decided that the three terminators should be varied to prevent off-target annealing of the overhangs during HiFi assembly.

The vector used for this construct was pUC19.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Stony_Brook--detectionplasmid.jpeg)

**Figure 7.** Final plasmid for the Detection system.

## References

Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J. W., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and Bioengineering, 114(12), 2828–2836. https://doi.org/10.1002/bit.26434

Dean, R. L. (2002). Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations. Biochemistry and Molecular Biology Education, 30(6), 401–407. https://doi.org/10.1002/bmb.2002.494030060138

Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001

Moore, C., Juan, J., Lin, Y., Gaskill, C., & Puschner, B. (2016). Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases. Marine Drugs, 14(3), 54. https://doi.org/10.3390/md14030054

Rosano, G. L., Morales, E. S., & Ceccarelli, E. A. (2019). New tools for recombinant protein production in Escherichia coli : A 5‐year update. Protein Science, 28(8), 1412–1422. https://doi.org/10.1002/pro.3668

## Anchoring Construct

The pKSI-1 vector was ordered from Addgene as an agar stab and was replated using the quadrant method. After overnight culture, colonies were inoculated in liquid Luria-Bertani (LB) media. After another overnight culture, we miniprepped using the Monarch Plasmid Miniprep kit to isolate the vector DNA for use in HiFi assembly.

For HiFi assembly, the vector was linearized by restriction enzyme digestion using the enzymes BamHI and HindIII. The NEBuilder HiFi DNA Assembly kit was used to ligate the DNA insert PgsA-MlrA with the vector, pKSI-1.

The plasmid was transformed into competent DH5-alpha *E. coli*, chosen because it can produce a high copy number of plasmids. NEB’s protocols for chemical transformation via heat shock were followed. Bacteria containing the plasmid were plated on agar plates with ampicillin. Once the colonies grew, some cultures were placed in liquid LB media and incubated overnight. Next, the cultures were miniprepped to isolate the final plasmid and nanodropped to ensure high DNA concentration.

![](https://static.igem.org/mediawiki/2021/1/1b/T--Stony_Brook--DH5alpha.jpeg)

**Figure 1.** Transformed DH5-alpha agar plate with anchoring plasmid.

Before transforming the final plasmid into BL21, it was important to ensure that the assembly step had proceeded as expected. Thus, a restriction enzyme digest was done using the enzymes SpeI and Kpn1. Since two Kpn1 restriction sites and one SpeI restriction site were present in the plasmid, three bands should appear on a gel. The size of the bands corresponded to the simulated agarose gel produced by SnapGene.

![](https://static.igem.org/mediawiki/2021/b/bf/T--Stony_Brook--Gel.jpeg)

**Figure 2.** Simulation of gel electrophoresis of the plasmid after RE digest (left) on SnapGene compared to actual gel electrophoresis of the plasmid after RE digest (right).

The final plasmid was chemically transformed using heat shock in competent BL21(DE3) and cultured on agar plates with ampicillin resistance.

![](https://static.igem.org/mediawiki/2021/e/e0/T--Stony_Brook--BL21plate.jpeg)

**Figure 3.** Transformed BL21(DE3) agar plate with anchoring plasmid.

## Testing the Anchoring Construct

An assay for MC-LR degradation was used to indirectly test for the presence of functioning MlrA protein. For this assay, the protocols adopted were based on work by Moore et al. and the 2014 Peking iGEM team, who also expressed MlrA and tested MC-LR degradation. This assay relies on the premise that MC-LR is an inhibitor of protein phosphatase 1 (PP1). PP1 catalyzes the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol (pNP), producing a yellow color. By monitoring absorbance at 405 nm in the presence of pNPP and PP1, the reaction rate, and indirectly the concentration of MC-LR, can be measured. High MC-LR concentration leads to greater PP1 inhibition and a less yellow solution (low absorbance). On the other hand, a low MC-LR concentration would be associated with less PP1 inhibition, faster conversion of pNPP to pNP and higher absorbance. If MlrA is correctly localized to the outer membrane, this would lead to lower MC-LR concentration, less PP1 inhibition and, in turn, increased absorbance.

A calibration curve was generated to determine the relative PP1 activity rate at MC-LR concentrations between 0 μg/L and 1 μg/L. At higher MC-LR concentration, the relative PP1 activity rate should decrease, as seen in the curve produced by Moore et al. However, in our attempts to generate this curve, the PP1 activity rate remained unchanged despite increasing microcystin concentration.

![Snow](https://static.igem.org/mediawiki/2021/5/53/T--Stony_Brook--litcalcurve.jpeg)

![Forest](https://static.igem.org/mediawiki/2021/8/8c/T--Stony_Brook--calcurve.jpeg)

**Figure 1.** Literature calibration curve from Moore et al. (top) compared to the generated calibration curve (bottom). Points on the right curve represent the average of three data points.

In order to indirectly test for MlrA expression, we hoped to use the PP1 assay to quantify how much microcystin remained after incubating transformed *E. coli* with MC-LR. We first incubated whole cells and cell lysate from our transformed BL21(DE3), as well as wild-type *E. coli*, with MC-LR at a final concentration of 100ng/L at 25 °C in accordance with the 2014 Peking iGEM team’s protocol. After 24 hours, we terminated the reaction by heating all solutions to 98 °C. Subsequently, we used the protocol from Moore et al. for the PP1 assay.

## Learning experience

Throughout this design cycle, there were some shortcomings that prevented the team from achieving ideal results. First, the transformed BL21(DE3) should have been tested for protein expression. Though RE digest and gel electrophoresis were conducted to check if the final plasmid contained the expected size insert, there was no guarantee that the *E. coli* would express MlrA, or that it would be anchored to the membrane. Our advisor, Dr. Gergen and teaching assistant Andrew Sillato, agreed that a western blot should have been performed to confirm PgsA-MlrA protein expression. However, this was not possible due to time constraints.

Also another issue our team faced was conducting the assay. As mentioned before, it was found that increasing microcystin concentration had no effect on PP1 activity, indicating an inactive inhibitor. In order to confirm this, it was decided to test our protocol with a different PP1 inhibitor. Therefore, we used sodium phosphate dibasic at varying concentrations in place of MC-LR, as inorganic phosphate is known to be an inhibitor of protein phosphatases (Dean, 2002).

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# Team:Stony Brook/Experiments

iGEM SBU 2021

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[Engineering Success](https://2021.igem.org/Team%3AStony_Brook/Engineering)
[Implementation](https://2021.igem.org/Team%3AStony_Brook/Implementation)

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[Parts Overview](https://2021.igem.org/Team%3AStony_Brook/Parts)
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[Integrated Human Practices](https://2021.igem.org/Team%3AStony_Brook/Human_Practices)
[Collaborations](https://2021.igem.org/Team%3AStony_Brook/Collaborations)
[Education & Communication](https://2021.igem.org/Team%3AStony_Brook/Communication)
[Partnership](https://2021.igem.org/Team%3AStony_Brook/Partnership)

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[Engineering Success](https://2021.igem.org/Team%3AStony_Brook/Engineering)
[Implementation](https://2021.igem.org/Team%3AStony_Brook/Implementation)

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[Parts Overview](https://2021.igem.org/Team%3AStony_Brook/Parts)
[Contribution](https://2021.igem.org/Team%3AStony_Brook/Contribution)

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Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3AStony_Brook/Human_Practices)
[Collaborations](https://2021.igem.org/Team%3AStony_Brook/Collaborations)
[Education & Communication](https://2021.igem.org/Team%3AStony_Brook/Communication)
[Partnership](https://2021.igem.org/Team%3AStony_Brook/Partnership)

![](https://static.igem.org/mediawiki/2021/4/4f/T--Stony_Brook--pacmanpreloader.gif)

# Experiments

## Protocol for PCR

### Materials:

* Q5 2X Master Mix: 12.5 uL
* 10 uM For Primer: 1.25 uL
* 10 uM Rev Primer: 1.25 uL
* DNA: 2.5 uL
* Nuclease free water: 7.5 uL
* Thermocycler
* PCR Tubes

### Preparing the thermocycler:

* Denaturation: 98 ℃; 5-10 s
* Annealing: 70 ℃; 10-30 s
* Extension: 72 ℃; 90 s (About 30 cycles)
* Final extension: 72 ℃, 180 s
* Nuclease free water: 7.5 uL

| Step | Directions |
| --- | --- |
| 1 | Start the annealing temperature at the higher Tm, go down 1 degree at a time for 1 cycle each until hit (lower Tm-5) and then back cycle that for 20 cycles. Start with 180s initial denaturation at 98 °C and end with 180s final extension at 72 °C. |

## Protocol for Agarose Gel Electrophoresis:

### Materials:

* Agarose
* Gel Tray
* Power Source
* Comb
* 1x TAE
* Ethidium Bromide

| Step | Directions |
| --- | --- |
| 1 | Weigh 0.5g agarose powder and 50ml 1X TAE buffer and add them to a flask. |
| 2 | Melt the mixture in a microwave until the solution becomes clear, and then add and mix EB or SYBR safe with the solution after the it cools to about 50~60 °C. |
| 3 | Pour the solution into the gel casting tray with appreciate comb. |
| 4 | Pull out the comb when the gel is completely solid. |
| 5 | Place the gel in the electrophoresis chamber and add enough TAE Buffer to submerge the gel. |
| 6 | Pipette DNA samples mixed with appreciate amount of loading buffer into wells on the gel. |
| 7 | Run the gel at 100V for about an hour. |

## Protocol for HiFi Assembly

### Materials:

* Ice
* DNA vector
* DNA fragment
* Pipette andTips
* Thermocycler
* NEB HiFi DNA Assembly Master Mix

| Step | Directions |
| --- | --- |
| 1 | Set up the following reaction on ice, according to [NEB:](https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol) ![](https://static.igem.org/mediawiki/2021/4/40/T--Stony_Brook--ExperimentNEB.jpeg) |
| 2 | Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases. |
| 3 | Transform NEB DH5-alpha or 10-beta Competent *E. coli* cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μl of the chilled assembled product, following the transformation protocol. |

## Protocol for NanoDrop

### Materials:

* NanoDrop
* Pipette
* Pipette Tips
* Tubes with prepared DNA
* Kimwipes

| Step | Directions |
| --- | --- |
| 1 | Make a blank measurement by pipetting 1µl of water onto the NanoDrop and press blank on the NanoDrop program. |
| 2 | Wipe the droplet of water |
| 3 | Pipette 1 µl of DNA solution, close the NanoDrop and press measure. |
| 4 | Record and label the results. |
| 5 | Repeat for all DNA samples. |

## Protocol for Competent Cells

### Materials:

* LB Media
* Agar plate
* Pipette
* Sterile 50 ml tube
* Centrifuge
* MgCl2

| Step | Directions |
| --- | --- |
| 1 | Streak out the *E. coli* strain on an LB plate to isolate colonies and incubate at 37 °C overnight. |
| 2 | Use a sterile pipette tip to collect cells from a single colony and inoculate in 5 ml sterile liquid LB Medium at 37 °C overnight in a shaker incubator. |
| 3 | Pipette 1 mL cells to 100 mL liquid LB Medium at 37 ℃ shaker incubator until grow the cultures to OD600 reaching about 0.26. |
| 4 | Pipette 25 ml of the cells to a pre-chilled sterile 50 ml tube. |
| 5 | Use a centrifuge to rotate at 4 °C, 4000 rpm for 10 minutes. |
| 6 | Decant supernatant and resuspend the cells by 10 ml ice cold 100 mM MgCl2. Hold on ice for 30 minutes. |

## Protocol for *E. coli* Transformation

### Materials:

* Competent Cells
* DNA plasmid
* Heat block and ice
* SOC media or LB media
* Agar Plates

| Step | Directions |
| --- | --- |
| 1 | Take competent cells out of -80°C and thaw on ice. |
| 2 | Warm up agar plates warm up to 37°C in the incubator. |
| 3 | Add of 2 μL vector into 50 μL competent cells, and GENTLY mix by flicking the bottom of the tube. |
| 4 | Incubate on ice for 20 min. |
| 5 | Heat shock each transformation tube in the 42°C water bath for 30 s. |
| 6 | Put the tubes back on ice for 2 min. |
| 7 | Add 950 μL SOC media or LB media(without antibiotic) to the bacteria and grow in a 37°C shaking incubator for 60 min. |
| 8 | Plate the transformation and incubate plates at 37°C overnight. |

## Protocol for Protein expression of BL21(DE3) using IPTG

### Materials:

* Transformed BL21(DE3) bacteria in LB media
* Pipette and Tips
* 100 mM stock solution of IPTG

| Step | Directions |
| --- | --- |
| 1 | The [NEB](https://www.neb.com/protocols/0001/01/01/protein-expression-using-bl21de3-c2527) protocol was followed. |

## Protocol PP1 Assay

| Step | Directions |
| --- | --- |
| 1 | Protocol from [Moore et al.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820308/) paper was followed. |

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# Team:Stony Brook/Results

iGEM SBU 2021

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# Results

## PP1 Assay

In order to indirectly test for MlrA expression, PP1 assay was used to quantify how much microcystin remained after incubating transformed *E. coli* with MC-LR. However, several issues arose when attempting to replicate the results from Moore et al. Firstly, the standard curve generated showed no inhibition by MC-LR, regardless of the concentration used. On the other hand, the curve from the literature demonstrated a clear decrease in relative PP1 activity with increasing MC-LR concentration. To determine whether lack of inhibition was due to the MC-LR solution used or a procedural error, it was decided to test another inhibitor of PP1. Given the limited time frame, it wasn’t possible to order a well-characterized, selective PP1 inhibitor, such as okadaic acid or tautomycin. Instead, 25mM dibasic sodium phosphate was used, since it is an inhibitor of other phosphatases and was readily available (Dean, 2002). However, unexpectedly, results from the PP1 assay demonstrated activation, not inhibition, in the presence of sodium phosphate (Figure 1). This is indicated by the fact that the log of the relative PP1 activity is positive and increases with increasing concentrations of sodium phosphate (as a percent of total reaction volume)—an inhibitor would produce negative values that increase in magnitude with greater inhibitor concentration.

![](https://static.igem.org/mediawiki/2021/e/e0/T--Stony_Brook--Figure1.jpeg)

**Figure 1.** Relative PP1 activity rate, expressed as log (exposed PP1 activity rate/control PP1 activity rate), for varying concentrations of sodium phosphate.

## Testing the Anchoring Construct

Despite the mixed results when generating the calibration curve, samples of transformed and wild-type bacteria were tested by the same protocol. Both cell lysate and whole cells were tested in order to get a sense for whether MlrA was present primarily on the outer membrane, as was hoped, or the cytoplasm, as in previous work (Dziga et al., 2012). However, as seen in Figure 2, these results proved to be inconclusive. While cell lysate of transformed BL21 did seem to result in reduced PP1 activity relative to the negative control (without sample exposure) at some concentrations, this was not true for all concentrations. Furthermore, these points did not show a strong correlation (R2=0.139). Similarly, data for whole cell samples from control and transformed *E. coli* had R2 values of 0.065 and 0.111, respectively. Given both the unsuccessful attempts at creating the calibration curve and the absence of strong correlations, it is unlikely that these results are an accurate representation of MlrA expression levels.

![](https://static.igem.org/mediawiki/2021/d/df/T--Stony_Brook--updatedfig2.jpeg)

**Figure 2.** Relative PP1 activity rate, expressed as log (exposed PP1 activity rate/control PP1 activity rate), for varying concentrations of sample.

## What we would do differently

Had we had more time, it would have been optimal to test the PP1 assay protocol with Phosphatase Inhibitor Cocktail 3 (produced by Sigma Aldrich), which is designed to be an effective inhibitor of PP1. It is composed of Cantharidin, (-)-p-Bromotetram­ isole, and Calyculin A. Then, we would have been able to definitively determine whether our MC-LR solution or protocol was at fault. Furthermore, in order to corroborate the results from the PP1 assay, a western blot for MlrA protein expression could have been conducted. Since MlrA contained a 6X His tag at the C-terminus, anti-His primary antibodies would be used for blotting. Alternatively, ELISA or LC-MS could have been carried out to determine the amount of microcystin present after incubation with samples.

## References

Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001

Dean, R. L. (2002). Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations. Biochemistry and Molecular Biology Education, 30(6), 401–407. https://doi.org/10.1002/bmb.2002.494030060138

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