iGEM 2020 — Team USAFA

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# Team:USAFA/Notebook

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* Poster

  [Poster](https://2020.igem.org/Team%3AUSAFA/Poster)
* Project

  [Description](https://2020.igem.org/Team%3AUSAFA/Description)
  [Design](https://2020.igem.org/Team%3AUSAFA/Design)
  [Experiments](https://2020.igem.org/Team%3AUSAFA/Experiments)
  [Parts](https://2020.igem.org/Team%3AUSAFA/Parts)
  [Attributions](https://2020.igem.org/Team%3AUSAFA/Attributions)
   [Contribution](https://2020.igem.org/Team%3AUSAFA/Contribution)
   [Implementation](https://2020.igem.org/Team%3AUSAFA/Implementation)
   [Model](https://2020.igem.org/Team%3AUSAFA/Model)
  [Notebook](https://2020.igem.org/Team%3AUSAFA/Notebook)
* Results

  [Results](https://2020.igem.org/Team%3AUSAFA/Results)
   [Engineering Success](https://2020.igem.org/Team%3AUSAFA/Engineering)
  [Future](https://2020.igem.org/Team%3AUSAFA/Future)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AUSAFA/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3AUSAFA/Education)
* Safety

  [Safety](https://2020.igem.org/Team%3AUSAFA/safety)
* Team

  [Team](https://2020.igem.org/Team%3AUSAFA/Team)
  [Collaborations](https://2020.igem.org/Team%3AUSAFA/Collaborations)
* Judging Form

  [Judging Form](https://2020.igem.org/Team%3AUSAFA/Judging%20Form)

>

# Notebook

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:USAFA/Engineering

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[![Logo](https://static.igem.org/mediawiki/2019/1/13/T--USAFA--logo.png)
![logo-mobile](https://static.igem.org/mediawiki/2019/1/13/T--USAFA--logo.png)](https://2020.igem.org/Team%3AUSAFA)

* Poster

  [Poster](https://2020.igem.org/Team%3AUSAFA/Poster)
* Project

  [Description](https://2020.igem.org/Team%3AUSAFA/Description)
  [Design](https://2020.igem.org/Team%3AUSAFA/Design)
  [Experiments](https://2020.igem.org/Team%3AUSAFA/Experiments)
  [Parts](https://2020.igem.org/Team%3AUSAFA/Parts)
  [Attributions](https://2020.igem.org/Team%3AUSAFA/Attributions)
   [Contribution](https://2020.igem.org/Team%3AUSAFA/Contribution)
   [Implementation](https://2020.igem.org/Team%3AUSAFA/Implementation)
   [Model](https://2020.igem.org/Team%3AUSAFA/Model)
  [Notebook](https://2020.igem.org/Team%3AUSAFA/Notebook)
* Results

  [Results](https://2020.igem.org/Team%3AUSAFA/Results)
   [Engineering Success](https://2020.igem.org/Team%3AUSAFA/Engineering)
  [Future](https://2020.igem.org/Team%3AUSAFA/Future)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AUSAFA/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3AUSAFA/Education)
* Safety

  [Safety](https://2020.igem.org/Team%3AUSAFA/safety)
* Team

  [Team](https://2020.igem.org/Team%3AUSAFA/Team)
  [Collaborations](https://2020.igem.org/Team%3AUSAFA/Collaborations)
* Judging Form

  [Judging Form](https://2020.igem.org/Team%3AUSAFA/Judging%20Form)

>

![](https://static.igem.org/mediawiki/2020/thumb/5/5e/T--USAFA--wikisideblock.jpg/43px-T--USAFA--wikisideblock.jpg)

# Engineering Success

![](https://static.igem.org/mediawiki/2020/thumb/6/6f/T--USAFA--engineering_success.png/584px-T--USAFA--engineering_success.png)

The USAFA iGEM team has worked diligently to achieve engineering success through the engineering design cycle in and out of the lab. Our efforts were inspired by the immense research the team has done on PFAS, to include its prevalence at many military bases and its hazards on human health. In fact, these military installations, including one local to USAFA, have run into the issue of costly and tedious methods to detect and degrade PFAS. The USAFA iGEM team imagined the immense benefits that would arise if they could generate a biologically synthetic solution, mitigating the prevalence of PFAS while cultivating a safer environment. Once the goal was set, the USAFA iGEM team has designed various experiments in search of potential methods of detection and degradation. These include observation of various dehalogenases, soil isolates, fungus isolates, winogradsky columns, and RFP detection methods, all of which served as potential solutions towards PFAS detection and degradation. We built each experiment by utilizing well-known techniques (plate streaking, microscopy and staining, fluoride ion probe, gel electrophoresis, etc.) and referencing scientific literature in order to test the intended goals for each observation source. WIth access to a laboratory, the USAFA iGEM team was able to test each experiment with its controls and various treatments to determine functionality and applicability to the project goals. Throughout the procedures, the team encountered numerous trial and error in which reiterations or revisions to tests were made in order to achieve the most reliant and relevant results. Overall, the team learned about various methods that can be used as approaches to the same goal, while also obtaining data that closely supports that the detection and degradation of PFAS is possible through these methods. Although the USAFA iGEM team’s results were not fully conclusive, the team plans to continue to conduct research and modify experimentation to seek solutions to the detection and degradation of PFAS.

## Associated Parts

![](https://static.igem.org/mediawiki/2020/thumb/d/dc/T--USAFA--pfascartoon2.png/800px-T--USAFA--pfascartoon2.png)1. [BBa\_K3347005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3347005)
2. [BBa\_K3347007](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3347007)
3. [BBa\_K3347009](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3347009)

| Part | Usage | Purpose | Creation |
| --- | --- | --- | --- |
| DeHa1.2.0 | PFOA/S destruction | Express Dehalogenase 1 variant 2 | DeHa1 in a T7 expression block with flanking Biobrick prefix and suffix as well as VF2/VR sites |
| DeHa1.2.HisN | PFOA/S Destruction | Express Dehalogenase 1 with N terminal His tag | DeHa1 in a T7 expression block with flanking Biobrick prefix and suffix as well as VF2/VR sites |
| DeHa1.2.HisCl | PFOA/S destruction | Express Dehalogenase 1 with C terminal His tag | DeHa1 in a T& expression block with flanking Biobrick prefix and suffix as well as VF2/VR sites |

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:USAFA/Experiments

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[![Logo](https://static.igem.org/mediawiki/2019/1/13/T--USAFA--logo.png)
![logo-mobile](https://static.igem.org/mediawiki/2019/1/13/T--USAFA--logo.png)](https://2020.igem.org/Team%3AUSAFA)

* Poster

  [Poster](https://2020.igem.org/Team%3AUSAFA/Poster)
* Project

  [Description](https://2020.igem.org/Team%3AUSAFA/Description)
  [Design](https://2020.igem.org/Team%3AUSAFA/Design)
  [Experiments](https://2020.igem.org/Team%3AUSAFA/Experiments)
  [Parts](https://2020.igem.org/Team%3AUSAFA/Parts)
  [Attributions](https://2020.igem.org/Team%3AUSAFA/Attributions)
   [Contribution](https://2020.igem.org/Team%3AUSAFA/Contribution)
   [Implementation](https://2020.igem.org/Team%3AUSAFA/Implementation)
   [Model](https://2020.igem.org/Team%3AUSAFA/Model)
  [Notebook](https://2020.igem.org/Team%3AUSAFA/Notebook)
* Results

  [Results](https://2020.igem.org/Team%3AUSAFA/Results)
   [Engineering Success](https://2020.igem.org/Team%3AUSAFA/Engineering)
  [Future](https://2020.igem.org/Team%3AUSAFA/Future)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AUSAFA/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3AUSAFA/Education)
* Safety

  [Safety](https://2020.igem.org/Team%3AUSAFA/safety)
* Team

  [Team](https://2020.igem.org/Team%3AUSAFA/Team)
  [Collaborations](https://2020.igem.org/Team%3AUSAFA/Collaborations)
* Judging Form

  [Judging Form](https://2020.igem.org/Team%3AUSAFA/Judging%20Form)

>

![](https://static.igem.org/mediawiki/2020/thumb/5/5e/T--USAFA--wikisideblock.jpg/43px-T--USAFA--wikisideblock.jpg)

# Experiments

## Dehalogenase

1. Transformed an D. Acidovorans plasmid with Ori.C, CAM (chloramphenicol resistance), Lac Operon and two types of DeHa3 dehalogenases as two separate variables into E.coli
2. The bacteria was grown overnight in LB broth and transferred to a test solution
   1. The culture required an aqueous solution to facilitate availability for free fluoride ion probe testing- There were a total of four different test solutions, each with varying concentrations of PFAS: 0, 100, 375 and 1500. All test solutions also contained chloramphenicol and Isopropyl β-d-1-thiogalactopyranoside (IPTG). All solutes were dissolved in LB broth. The different tests are shown below.
     1. Negative Control: DE3 690 without IPTG (not a required activator for DE3) expressing nucleocapsid protein from SARSCoV2, which should not affect fluoride ion concentration
     2. Negative Control: DeHa3 with Delta 1 dehalogenase without IPTG, a necessary activator to conduct dehalogenase function
     3. Positive Control: Known fluoride ion solution
     4. DeHa3 with Delta 1 dehalogenase with IPTG
     5. DeHa3 with Delta 2 dehalogenase with IPTG- Cells were sonicated after 24 hours of growth in test solution
     - At 0, 6 and 24 hours, free fluoride ions were measured using a free fluoride ion probe
     1. If there is an increase in measurements, then a pattern of dehalogenase activity would be present

## Fungus Isolate

### Winogradsky Sampling

1. 0.1% PFOA and Agar minimal media plates were made
2. All winogradsky column samples were streaked on the minimal media plates
3. Plates were incubated at 30 degrees Celcius for 14 days and observed daily
4. Upon observing a plate with positive growth, the fungal growth was transferred to a SAB plate
5. Lastly, once fungus/fungi are grown on a positive media, they are sent out for sequencing

![](https://static.igem.org/mediawiki/2020/thumb/e/e7/T--USAFA--rainbowfungusv2.jpg/675px-T--USAFA--rainbowfungusv2.jpg)

### Schriever AFB Contaminated Soil Sampling

1. Add contaminated soil and DI water into an eppendorf tube
2. Vortex until fully mixed and let the sediment settle
3. Pipet 200 μL of the mixed DI on top of the sediment was put over a minimal media plate of PFOA (0.1%) and Agar and was spread
4. Leave the plate out for 10 days at room temperature
5. Transfer positive growth to a SAB plate
6. Send fungus for sequencing

![](https://static.igem.org/mediawiki/2020/thumb/f/f6/T--USAFA--fungus2.jpg/675px-T--USAFA--fungus2.jpg)

For both the Winogradsky sampling and contaminated soil sampling, slants were created of the same PFOA agar minimal media to test for for growth from SAB grown fungus to minimal media again. (all fungus we grew were able to regrow on the minimal media)

## Winogradsky Community

1. Three overnight cultures were made for Delftia *acidovorans*, Pseudomonas *flourescens* and E. *coli* in 1/3 conc. TSB
2. Three mL of bacterial sample transferred into each vial
3. Centrifuged each vial to form bacterial pellet
4. Removed supernatant
5. Added 3mL of either DI water, 1000ppm PFOA, or 500ppm PFOS

1. Negative controls: just 1000 PPM PFOA, just 500 PPM PFOS, and the bacterial species in DI water

6. Vortex viles to recombine in solution
7. Place vials on ice
8. Half of the samples were sonicated to lyse the cells. Half were left with whole cells.
9. Once sonication was complete, the fluoride ion level was measured in each vial at 0, 1, 2, and 24 hrs.

## Detection

1. Searching for constitutive genes within the rhodococcus genome which constitutively/ housekeeping genes = want to show system works/ positive control, chose glyA,16S, recA
2. See if can activate prmA in rhodococcus has been shown to upregulate in rhodo in presence of PFAS
3. Evidence of some dehalogenase capability to break down PFOS, wanted to see if promoters would be activated (might degrade, so might be something that promotes degradation pathway)
4. Flanked promoter with EcoR1 and Not1 cut sites, cut out the Lac promoter region of plasmid pSRKBB using EcoR1 and Pst1 restriction enzymes so only have ORI and kanamyasin resistance gene in order to insert in RFP
5. Ran gel to check bp length for promoters and pSRKBB
6. Purify DNA using gel extraction protocol
7. Ligate RFP sequence into BB plasmid to be used as reporter gene with multi-cloning site immediately upstream of RFP to insert promoters
8. Transform ligated plasmid (BB with RFP) into E. coli
9. Grow up colonies
10. Tested 5 colonies by diagnostic restriction enzyme digest, to cut same fragment out and determine which colonies had fragment inserted using miniprep
11. 3 out of 5 colonies had correct size fragment for RFP
12. Once found colony that worked, did miniprep on DNA cut it with different set of restriction enzymes, which allow to ligate promoter sequences immediately upstream of RFP at Not1 site
13. Grew up on E. coli
14. Future action:

1. Check for consecutive promotion
2. prmA/Dehalogenase to turn red with PFAS transformed

## Basic Lab Prep and Growth Procedures

![](https://static.igem.org/mediawiki/2019/5/54/T--USAFA--experiments1.jpg)

### Making LB

1. Read the directions on the bottle . For example, to make LB Agar. The bottle says to add 40 grams off the LB powder per liter of distilled water. (Note: the gram amount may differ for each bottle. See bottle for instructions).
2. Scale 12.5 grams of LB powder of 500 mL of distilled water with graduated cylinder.
3. Always heat the mixture to dissolve fully using a heat plate and a flask. Use an autoclaved flask. Make sure to have stir bir to dissolve particles. Black autoclaved tape on the flask means that it has been autoclaved and is sterile. Temperature doesn't matter. Dark urine color.
4. Autoclaved for 15 minutes in 121 degrees. TIghten media bottle cap . Put foil on top of lid and put autoclave tape. Use double volume bottle for however much liquid mixture being autoclaved. For example, use a liter bottle for a 500 mL mixture.
5. Tip when labelling with masking tape, flip end of one tape so easy to pull off.
6. Always triple rinse glassware with DI water after washing with soap and warm water.

![](https://static.igem.org/mediawiki/2019/f/f9/T--USAFA--experiments2.jpg)

### Bacterial Plating

1. Add 5 mL of LB broth to each test tube (cultured tubes) (3 of them).
2. From the bacteria plate, take a distinct colony for each test tube and swirl it into the LB broth.
3. Plate two more plates with the original plate. Streak on one side of the plate, do a quarter turn, and then overlap the streaks at this direction.
4. Placed the plates in the incubator and ensure that all surfaces are cleaned. The temperature will vary based on different bacteria.

### Making Gel for Electrophoresis

![](https://static.igem.org/mediawiki/2019/1/13/T--USAFA--experiments3.jpg)

#### (For 50ml of 2% agarose gel)

1. Add 1 gram of iGEM agarose (B/c 2% of 50 is 1)
2. Add 50 ml of 1x tbe ( a bit more over 50 is okay due to evapoation) and mix
3. Stuff flask with tissue
4. Microwave for 30 seconds- stop when mix starts to boil to avoid over flowing, start again when solution settles
5. OPTIONAL, either mix in GelGreen or GelRed, or dye with EtBr later after Electrophoresis ran.
6. Continue to microwave until there are no more agarose crystals left. Make sure the ENTIRE solution is clear, Look meticulously.
7. Once it is clear then it can be poured.
8. Let it cool in the fridge for about 10mins, or until opaque.

### Gel Preparation(Summer procedure)

![](https://static.igem.org/mediawiki/2019/5/5a/T--USAFA--experiments4.jpg)

#### Make a gel buffer, then add agarose.

1. Obtain a 1 liter container. Add 20 mL of buffer and fill the rest of the container with 18 miliohm water until 1 liter volume is attained.
2. Pour 50 mL of the solution into a smaller flask. Add 0.4 g of agarose powder. Microwave mixture for about a minute and a half (you can allow it to boil for 20-30 sec).
3. When the flask can be held comfortably for 5-6 sec, add 5 microliters of ethidium bromide and swirl to mix. Pour mixture into gel container and insert the comb (this makes the wells) onto the side where the DNA and ladders will be inserted. The gel will solidify in about 20 min.
4. Pull comb out of solidified gel. Plug in power source and hook wires to gel electrophoresis instrument ensuring that the DNA will flow from negative to positive.
5. Pour buffer into gel electrophoresis instrument so that a thin layer covers the top of the gel.
6. Add 5 microliters of each sample, keeping track of what well each sample is insterted into.
7. Set power to 130-140 V for about a half hour or until the DNA has moved about halfway or 3/4 of the way down the plate.

![](https://static.igem.org/mediawiki/2019/1/16/T--USAFA--experiments6.jpg)

### How to Grow & Preserve *Rhodococcus RHA1* in Glycerol for Freezing

1. Culture bacteria in LB broth in incubating orbital minishaker at 30 degrees C and 250rpm for 48 h.
2. Under biosafety cabinet (hood), add 500 mL of *Rhodococcus RHA1* in broth to Corning cryotube
3. Under hood, add 500mL 50% sterile glycerol mix (See below if you need to make this)
4. Place tubes into cryo Freezing Container
5. Place in -80 Freezer
6. Next day, take out of -80 freezer and put into a box in the -80 freezer.

## DNA+RNA Prep

### Digestion

![](https://static.igem.org/mediawiki/2019/7/7f/T--USAFA--experiments9.jpg)

1. For digestion, use 1-5 micrograms of DNA, buffer at 10x concentration
2. 1 unit of enzyme per microgram of DNA that can be digested in 1 hour
3. Fill the rest of the desired volume with 18 miliohm water.
4. Total volume shouldn't exceed 60 microliters.

### Tradition Ligation

1. 7 ul vector (~10.7 ng/ul)
2. 10 ul Insert (`10 ng/ul)
3. 1 ul Enzyme (T4)
4. 2 ul Buffer (T4)
5. Mix all four together (in order of descending volume) then let sit at room temp (or 16 degrees celsius) overnight.

### qRT-PCR

![](https://static.igem.org/mediawiki/2019/5/59/T--USAFA--experiments10.jpg)

1. For each sample, weigh 25–50 mg acid-washed glass beads (150–600 μm diameter) in a 2 ml Safe-Lock tube (not supplied) for use in step 4.
2. Harvest bacteria by centrifuging at 5000 x g for 5 min at 4ºC (do not use more than 1 x 109 bacteria). Decant the supernatant, and carefully remove any remaining media by aspiration. If the centrifuge will be used later in this procedure, heat it to 20–25ºC. Note: Incomplete removal of medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.
3. Add the appropriate volume of Buffer RLT (see table below). Vortex vigorously for 5– 10 s. 9. Note: Ensure that β-ME is added to Buffer RLT before use (see “Important points before starting”). Ensure that the pellet is thoroughly resuspended in Buffer RLT
4. Transfer the suspension into the 2 ml Safe-Lock tube containing the acid-washed beads prepared in step 1. Disrupt cells in the TissueLyser for 5 min at maximum speed. Other methods of mechanical disruption can be used instead. We recommend using the TissueLyser
5. Centrifuge for 10 s at maximum speed. Transfer supernatant into a new tube (not supplied). The volume of the tube must be at least twice that of the Buffer RLT used.
6. Determine the volume of supernatant. Add an equal volume of ethanol (70%), and mix well by pipetting. Do not centrifuge. After adding ethanol, a precipitate may form. This will not affect the RNeasy procedure.
7. Transfer up to 700 μl lysate, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.\* Reuse the collection tube in step 8. If the lysate exceeds 700 μl, centrifuge successive aliquots through the spin column. Discard the flow-through after each centrifugation.\*
8. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.\* Reuse the collection tube in step 9. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely. Skip this step if performing optional on-column DNase digestion.
9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flowthrough. Reuse the collection tube in step 10. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Important points before starting”).
10. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
11. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 10.
12. If the expected RNA yield is >30 μg, repeat step 12 using another 30–50 μl RNase-free water, or using the eluate from step 12 (if high RNA concentration is required). Reuse the collection tube from step 12. If using the eluate from step 12, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.

![](https://static.igem.org/mediawiki/2019/5/52/T--USAFA--experiments11.png)

### QIAquick PCR Purification

1. Used for purification of up to 10 ug of PCR product (10 - 100 bp) - (Used for G block)
2. Add 5 volumes of PB Buffer to 1 volume of PCR product and mix. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
3. place a QIAquick column in provided 2 ml collection tube.
4. To bind DNA apply sample to QUIquick column (centrifuge at 13000 rpm for 30-60 second). Discard flow through and insert column back in the same tube
5. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
6. Centrifuge QIAquick column once more in provided 20 ml collection tube to remove residual wash buffer.
7. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
8. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
9. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

![](https://static.igem.org/mediawiki/2019/e/e6/T--USAFA--experiments12.jpg)

### QIAquick Gel Extraction Kit

1. Used for purifying of up to 10 ug of DNA from gel.
2. Cut the DNA from the agarose gel
3. Weigh gel in colorless tube. Add 3 volumes QC Buffer per 1 volume gel (gel ~ 100 mg = 10 ul).
4. Incubate tube 50 - 60 degrees for 10 minutes (or until gel completely dissolved). Vortex every 2 - 3 minutes to break down gel. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
5. Place sample in 2 ml collection tube. Centifuge for 1 minute or until all sample passes through column. Discard flow through.
6. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
7. Repeat step 5
8. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
9. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
10. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

### MINIPREP

![](https://static.igem.org/mediawiki/2019/e/e8/T--USAFA--experiments13.jpg)

1. Pellet 5 mL of culture into each Epindorf tube, 6 tubes total. Add 2 mL at a time, only adding 1 mL in the last addition. Spin down tube in centrifuge after each 2 or 1 mL addition.
2. Resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
3. Add 250 microliters of lysis solution to each sample; invert 4 times to mix.
4. Add 10 microliters of the protease solution to the sample; invert 4 times to mix. Incubate at room temperature for 5 min.
5. Add 350 microliters of Neutralization Solution; invert 4 times to mix.
6. Centrifuge at top speed for 10 minutes at room temperature.
7. Insert spin column into collection tube.
8. Decant cleared lysate into Spin Column.
9. Cetrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.
10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
11. Repeat step 10 with 250 microliters of wash solution.
12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
13. Transfer spin column to a 1.5 mL microcentrifuge tube.
14. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution.
15. Discard column and store DNA at -20 deg C. If in multiple tubes, combine liquid from all tubes into one.

### MINIPREP (promega)

![](https://static.igem.org/mediawiki/2019/c/cc/T--USAFA--experiments14.jpg)

1. Pellet 8 mL of culture into 5 Epindorf tube, using an additional counterbalance tube (6 tubes total). Add 2 mL at a time, then spin at 14000 rpm for 1 minute after each. (Add 2 mL, spin, dump, add 2 more.. etc). After last spin, decant lysate and remove any remaining fluid near the pellet with a micropipette.
2. Thoroughly resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
3. Add 250 microliters of lysis solution to each sample; forcibly invert 4 times to mix.
4. Add 10 microliters of the protease solution to the sample; forcibly invert 4 times to mix. Incubate at room temperature for 5 min.
5. Add 350 microliters of Neutralization Solution; forcibly invert 4 times to mix.
6. Centrifuge at top speed (1400 rpm) for 10 minutes at room temperature.
   Binding of Plasmid DNA
7. Insert spin column into collection tube.
8. Decant cleared lysate into Spin Column.
9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.

##### Washing

10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
11. Repeat step 10 with 250 microliters of wash solution.
12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
13. Transfer spin column to a 1.5 mL microcentrifuge tube.
14. Let column sit 3-5 minutes to allow ethanol to evaporate out.
15. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution.
    -Discard columns (keep flowthrough) and digest samples with Eco RI.

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# Team:USAFA/Results

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* Poster

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* Project

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>

# Results

## Microbial Library Screening

Individual colonies of each bacterial species were taken from TSA (tryptic soy agar) plates and grown in TSB (tryptic soy broth) overnight to ensure cells were viable. Cells were pelleted at 4500xg for 5 min and suspended in phosphate buffered saline (PBS; pH 7.4) and pelleted again to wash the pellet. The pellet was then suspended in Raymond’s minimal media with no additional carbon source added. This suspension was used to inoculate the following experimental samples:

* Raymond’s media + no carbon: negative growth control
* Raymond’s media + 1 PPM PFAS (PFOA or PFOS): test sample
* Raymond’s media + 100 PPM glucose: positive growth control
* Raymond’s media + 1PPM PFAS (PFOA or PFOS) + 100 PPM glucose: toxicity control

Following inoculation, samples were monitored daily/bi-weekly for optical density as a measure of growth for approximately 1 month.

The experiment was setup in such a way that if any particular sample showed no growth in the negative control, and positive growth in all remaining samples, it could be concluded that the added PFAS was likely being used in some way in the cellular metabolism. The toxicity control was included to ensure that the sample was not failing to grow simply due to PFAS cytotoxicity.

It was found for a majority of the samples, either a significant decrease in OD was observed when exposed to PFAS as a sole carbon resource, or no significant OD increase was observed compared to the negative control. A single sample did show a very small OD increase when grown in 1PPM PFOA, along with acceptable results for the controls (no growth in negative control, very strong growth in controls containing glucose). This sample was identified as Delftia acidovorans, and was used in further studies.

## *Delftia acidovorans* Fluoride Studies

In order to determine if this sample was capable of defluorinating PFOA, a fluoride monitoring experiment was performed. Delftia acidovorans was grown in 1 PPM PFOA in an identical fashion to the previous experiment. A larger scale was used to allow frequent aliquot removal to measure fluoride content by selective ion probe. This measurement was performed by removing 1 mL aliquots from growth mixture, and pelleting of cells by centrifugation. The aliquot was then tested directly by the probe for fluoride content. A comparison was performed by growing Pseudomonas fluorescens (a common gram negative soil bacteria) with a similar compound, ethylflouroacetate (EtFA) at 1 PPM. This compound has been shown previously to degrade by defluorinating strains such as Pseudomonas fluorescens. Thus, this serves as a positive control for our measurements.

![](https://static.igem.org/mediawiki/2020/6/61/T--USAFA--results_fig_1.png)Figure 1: Fluoride release compared to growth of Delftia acidovorans , measured by OD600. Growth was performed in Raymond’s minimal media with 1PPM PFOA.

![](https://static.igem.org/mediawiki/2020/a/ac/T--USAFA--results_fig_2.png)Figure 2: Fluoride release compared to growth of Pseudomonas fluorecens, measured by OD600. Growth was performed in Raymond’s minimal media with 1PPM EtFA.

As shown in Fig 1, D. acidovorans does exhibit growth in minimal media with PFOA as the sole carbon resource. However, no changes in fluoride concentration could be seen over the course of the experiment, suggesting that either the fluoride concentration was below the limit of detection of the probe, or metabolism was limited to the terminal carbon of PFOA, where no fluorine is present. Fig. 2 shows that our measurement method does work as expected, as fluoride concentration increases along with the increase in OD for P. fluorescens.

## *Delftia acidovorans* Cell Lysis Studies

To test the possibility that enough fluoride was not liberated to stand out from the background in the previous experiment, a follow up was performed that eliminated monitoring cellular growth. Instead large amounts of cells were grown in TSB and lysed to expose the cytosol content. This crude lysate was then exposed to either PFOA (100 PPM), EtFA (100PPM), or water and fluoride concentrations were monitored over time using the selective ion probe (Figure 3). A positive control using P. fluorescens was run under identical conditions (Figure 4).

Cell lysis was performed by sonication of cell pellets suspended in PBS. In addition, half of the samples were not lysed, and are referred to as cell slurries for this experiment.

![](https://static.igem.org/mediawiki/2020/e/ef/T--USAFA--results_fig_3.png)Figure 3: Fluoride measurement of D. acidovorans crude lysates. Data collected at each time point was linearly scaled such that the negative control (water) was identical to the starting conditions to account for possible fluctuations not associated with defluorination.

![](https://static.igem.org/mediawiki/2020/1/17/T--USAFA--results_fig_4.png)Figure 4: Fluoride measurement of P. fluorescens crude lysates. Data collected at each time point was linearly scaled such that the negative control (water) was identical to the staring conditions to account for possible fluctuations not associated with defluorination.

In both cases, fluoride release was observed in both P. fluorescens and D. acidovorans when exposed to PFOA when cells were lysed. A very small increase in fluoride was observed for PFOA in the cell slurries for both samples as well. Interestingly, it was observed that the fluoride concentrations in the cell lysates did not maintain over time, as seen by the decrease in signal after a peak value. This was hypothesized to be due to fluoride being incorporated into other molecules, resulting in loss of signal. It would be expected that any protein activity in a crude lysate would decrease as active peptidases would be functional in this reaction setup. Therefore we assumed that any defluorinating enzyme was being destroyed throughout the reaction, and that the rate of fluoride production in the system was decreasing until it fell below the rate of fluoride uptake in side reactions, leading the data seen in this experiment.

At this point, we decided to attempt to identify a possible dehalogenase from Delftia acidovorans. The genome of this microbe is known, and two haloacid dehalogenases were identified (PZP66635.1& WP\_011137954.1), referred to as type I and type II. Both sequences were used in modeling using Phyre as well as used to construct biobricks for testing in E. coli.

## *Delftia acidovorans* Dehalogenases

### Naming Scheme

Naming notation for Dehalogenase genes and expression blocks

*Plasmid notation*:
Represented by pXX\_DeHa
Where XX can represent:
C: pSB1C3
A: pSB1A3
K: pSB1K3
KB: pSRKBB

*Dehalogenase gene & Expression Block Notation*
Represented by DeHaX.Y.Z
Where,
X is the gene type (1 or 2 ; referencing the two dehalogenase genes in *D. acidovorans*)
Y is the variant (native variant = 1, *E. coli* codon optimized = 2, etc.)
Z shows the tags/modifications on the protein (0 = no modifications, HisC = 6xHis tag on carboxyl terminal, HisN = 6xHis tag on amine terminal, gene, etc.)
***Note:*** If Z is replaced by the 'gene' modifier, the Dehalogenase gene sequence is being referenced. In all other cases, the fully assembled expression block is referenced. Example: DeHa1.1.gene references BBa\_K3347000 while DeHa1.1.0 references BBa\_K3347001.

*Example notations*
**pKB\_DeHa2.2.HisN**: Dehalogenase type II, variant 2 (*E. coli* optimized) with a N terminal 6xHis tag all inside the pSRKBB vector
**pC\_DeHa1.1.0**: Dehalogenase type I, variant 1 with no further tags or modifications all inside the pSB1C3 vector

### Expression in *E. coli*

We expressed several variants of the dehalogenase genes in E. coli, including the native Dehalogenase type I and II under the control of the lac promoter in DH5a, as well as the E. coli optimized variants for both types I and II under the control of the T7 promoter in BL21 DE3. Among these optimized variants, His tagged versions were also expressed, ultimately leading to the expression of the following proteins in E. coli:

1. pC\_ DeHa1.1.0 in DH5a
2. pC\_ DeHa2.1.0 in DH5a
3. pKB\_DeHa1.2.0 in BL21 DE3
4. pBK\_DeHa1.2.HisC in BL21 DE3
5. pBK\_DeHa1.2.HisN in BL21 DE3
6. pBK\_DeHa2.2.0 in BL21 DE3
7. pBK\_DeHa2.2.HisC in BL21 DE3
8. pBK\_DeHa2.2.HisN in BL21 DE3

Each of these strains were grown in 50 mL LB + appropriate antibiotic selector at 37 degrees C in a shaking incubator until OD reached 0.6, at which point cultures were split into 2x25mL aliquots. One aliquot received IPTG (1mM) to induce protein production. Cultures were shaken for an additional 6 hrs before harvesting. E. coli were lysed by pelleting at 5000xg for 10 min, and suspending in lysis buffer (PE LB from G-Biosciences + C0mplete protease inhibitor cocktail + 0.4 mg/mL Lysozyme), and shaken for 1 hr at 37 degrees C. Soluble proteins were separated from crude cell extract by centrifugation (5000xg for 30 min at 4 degrees C) and transferred to clean micro-centrifuge tube. Soluble protein extracts were placed on ice when in use and frozen at 0C for storage.

### Initial Analysis of DeHa1.2 and DeHa2.2

To determine if catalytic activity is seen on small scale, a simple experiment was devised to monitor fluoride as a degradation product of PFAS hydrolysis. To a reaction vessel a sample of soluble protein extract was added to either PFOA, EtFA, or water. A time 0 measurement was made immediately upon addition and fluoride monitored over the course of 200 minutes. REsults can be seen below in figure 5. From this simple experiment it appears that both the positive control, EtFA, as well as PFOA-containing samples exhibit rapid fluoride concentration increase followed by a large period of unchanging concentrations when the blank was subtracted. The blank (not shown) exhibited a small increase in fluoride upon addition to a much lower extent than either samples. A cause of concern is the abundance of fluoride released throughout the course of the experiment. No greater than 400 ppb fluoride was measured in any sample, suggesting that while defluorination is occuring, the equilibration point is very low, ultimately resulting in very little free fluoride in solution. This analysis is not fully conclusive, and our team is not comfortable at this time making the claim that we have identified a PFAS defluorinating enzyme; however we are currently working to optimize reaction conditions for these enzymes in order to perform more in-depth analysis. Please reference our future work page for more detail.

![](https://static.igem.org/mediawiki/2020/8/82/T--USAFA--Fluoride_concentration_monitor_PFOA_ETFA_DeHa1_2_2.png)

Figure 5: Time-correlated fluoride concentration monitoring of A: DeHa1.2 and B: DeHa2.2 upon exposure to PFOA and EtFA. Fluoride concentration was measured by a selective ion probe.