iGEM 2018 — Team WPI_Worcester

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# Team:WPI Worcester/Notebook

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# Notebook

* [Protein Purification Notebook](https://static.igem.org/mediawiki/2018/f/f5/T--WPI_Worcester--Bailey_Notebook.pdf)
* [Antifreeze Protein Notebook](https://static.igem.org/mediawiki/2018/7/77/T--WPI_Worcester--Antifreeze_Protein_Notebook.pdf)
* [Lettuce Notebook 1](https://static.igem.org/mediawiki/2018/a/a2/T--WPI_Worcester--Lettuce_Notebook_1.pdf)* [Lettuce Notebook 2](https://static.igem.org/mediawiki/2018/f/fe/T--WPI_Worcester--Lettuce_Notebook_2.pdf)


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# Team:WPI Worcester/Experiments

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## LB Media

### Material

* 2 Liter Flask
* LB Broth Powder
* DI Water
* Bottles
* Stir Bar
* 1 Liter Graduated Cylinder
* Balance
* Cup

### Protocol

1. Place a large stir bar in a 2 liter flask
2. Place the 2 liter flask on a stir plate
3. Use a 1 liter graduated cylinder to measure 1 liter of DI water and pour it into the 2 liter flask
4. Turn on the stir plate
5. Place a cup on a balance and tare the balance
6. Use a scoop to measure out 40 grams of LB Broth powder
7. Carefully add the 40g of LB broth powder to the 2 liter flask
8. Allow to mix until all of the powder is dissolved
9. Pour The LB into smaller (250mL) bottles
10. Leave caps slightly loose and tape with autoclave tape
11. Autoclave the LB
12. Tighten caps

## LB Agar

### Materials

* 2 Liter Flask
* Stir plate
* Petri Dishes
* 50℃ Water Bath
* Bunsen Burner
* Paper Towel
* Colored Sharpie
* Tinfoil
* DI Water
* Balance
* Cup
* Stir bar

### Protocol

1. Tare the balance with a cup on it
2. Measure 40 grams of LB Agar into the cup
3. Put 1 liter of water in a 2 liter flask on a stir plate
4. Add the LB agar into the flask and a stir bar into the flask
5. Allow to mix until powder is dissolved
6. Put tinfoil on the top of the flask and add a strip of autoclave tape
7. Autoclave the flask
8. Mix the flask again
9. Place the flask in 50℃ water bath for 30 minutes to 2 hours
10. Add antibiotic to the flask (1mL to 1L)
11. Prep empty petri dishes in stack of 5 for easy pouring
12. Label petri dishes with appropriate color that corresponds to antibiotic
13. When ready to start pouring, light the bunsen burner and retrieve the flask from the water bath
14. Wrap a paper towel around the neck of the flask to avoid it getting slippery while pouring
15. Before pouring each stack of 5 petri dishes, place the lip of the flask into the flame of the bunsen burner to ensure it is sterile
16. Pour all of the plates
17. Let the plates cool overnight
18. Bag the plates and store in the fridge for up to a few months

## DNA Miniprep Materials * A1 buffer * A2 buffer * A3 buffer * A4 buffer * Elution buffer * Overnight culture of E. Coli * Column * Microfuge tube Protocol 1. Prepare an overnight culture of the E. coli with desired plasmid (5mL LB + 1 colony + 5uL of desired antibiotic) 2. Incubate in shaker at 37℃ overnight 3. Spin down culture in big centrifuge at 3500 rpm for 10 minutes 4. Dump out supernatant into waste beaker 5. Resuspend pellet in 250 uL A1 buffer 6. Move culture into 1.5mL microfuge tube 7. Add 250 uL A2 buffer 8. Incubate for 5 minutes (mix well, should be blue and sticky) 9. Add 300uL of A3 buffer (mix until white and fluffy) 10. Spin in small centrifuge at max speed for 10 minutes 11. Set up and label columns while sample is spinning 12. Move supernatant into the column 13. Spin in small centrifuge 30 seconds max speed 14. Dump liquid 15. Add 600uL of A4 16. Spin for 30 seconds at max speed 17. Dump liquid 18. Spin for 2 minutes 19. Move column into microfuge tube 20. Add 50uL elution buffer 21. Spin 1 minute at max speed 22. Nanodrop to check yield 23. Plug in the nanodrop 24. Wash the metal plates with DI water 25. Dry with kimwipe 26. Put 2uL elution buffer onto bottom plate 27. Run blank 28. Confirm blank by running elution buffer again 29. Wipe off plates and then run samples 30. 2uL of sample and record the ng/uL

## Transformations

### Materials

* Competent cells
* 1.5mL microfuge tubes
* Plasmid DNA
* Ice bucket
* Water Bath (42℃)
* SOC Medium
* 37℃ shaker
* Agar plates
* Incubator

### Protocol

1. Thaw competent cells on ice (~30 minutes)
2. Pre-chill 1.5mL microfuge tubes
3. Add 50 uL of competent cells into each microfuge tube
4. Add 1uL of plasmid DNA into the tubes
5. Incubate on ice for 30 minutes
6. Heat shock in a water bath (42℃) for 45 seconds
7. Incubate on ice for 5 minutes
8. Add 250uL of SOC medium to each tube
9. Incubate in shaker at 37℃ for 1 hour
10. Plate the 250uL of cells onto agar plates
11. Incubate overnight

## NCTC Electroporation

1. Autoclave 500mL of LB in a 1 liter flask
2. Autoclave ~2000mL of DI water (then put in 4℃ fridge)
3. Make 50mL of overnight culture
4. Let incubate overnight
5. Add the 50mL overnight culture to 500mL LB in flask
6. Put in 37℃ shaker until the OD is at ~0.4
7. Centrifuge in big centrifuge at 3500rpm for 10 minutes at 4℃
8. Discard supernatant (dump into waste and tap on paper towel)
9. Resuspend in 500mL of ice cold ddH2O and spin and dump
10. Do this rinse three times at least
11. Resuspend in 1mL Ice cold 10% glycerol
12. Make 100uL aliquots (Keep in -80℃ freezer or on ice if electroporation is happening directly after)
13. Chill 1.5mL microfuge tubes
14. Add 50uL of cells to the microfuge tube
15. Add 1uL of plasmid DNA to the microfuge tube
16. Incubate on ice for 10-30 minutes
17. Carefully put into the electroporation cuvette
18. Get volume to 100uL using ddH2O
19. Put cuvette in electroporation apparatus
20. 2-5 kilovolts
21. Add 950uL SOC medium to the electroporation cuvette to rinse the cells out of cuvette
22. Move cells into 15 mL tube and incubate for 1 hr at 37℃
23. Plate the cells on agar plates with appropriate antibiotic

## M9

### M9 Salts

1. 800mL H2O
2. 64g Na2HPO4-7H2O
3. 15g KH2PO4
4. 2.5g NaCl
5. 5g NH4Cl
6. Stir until dissolved
7. Adjust to 1000mL with distilled H2O
8. Separate into 5 200mL aliquots
9. Autoclave to sterilize

### M9 Media

1. 700mL ddH2O
2. 200mL M9 Salts (above)
3. 2mL MgSo4 (sterile)
4. 20mL of 20% glucose (or 20% glycerol)
5. 100uL of 1M CaCl2
6. 1mL of sterile, filtered 1000x L-Arginine
7. Adjust to 1000mL using ddH2O

## Mg Chemically Competent Cells

### Materials

* 15mL conical tubes
* 1.5 mL microfuge tubes
* CCMB80 buffer
* Overnight culture
* Centrifuge

### Protocol

1. Prepare an overnight culture (x4) (5mL LB + 1 colony)
2. Incubate overnight
3. Spin overnight culture for 10 minutes at 3500rpm
4. Pour supernatant into waste beaker
5. Resuspend in 80mL of CCMB80 buffer (20 in each tube)
6. Incubate on ice for 20 minutes
7. Spin for 10 minutes at 3500rpm
8. Resuspend in 10mL CCMB80 buffer (2.5 in each tube)
9. Combine cells from all tubes
10. Incubate on ice for 20 minutes
11. Aliquot 400uL into each microfuge tube
12. Store at -80℃ and discard thawed cells

## Protein Purification

### Materials

* Lysis Buffer
* Equilibration Buffer
* Wash Buffer
* Elution Buffer
* BL21 Cultures of GFP, IA, and ZeAFP
* Tissue Grinder
* NiNTA Slurry

## Protocol

1. Add 5mL of LB, 5uL of AMP, and one picked colony from the BL21 strains and grown overnight
2. Colonies grown overnight
3. 1 mL of culture were added to 200mL of LB
4. OD was checked. Goal was to be between 0.05-0.1 before incubation
5. After incubation for 3 hours, the move the cultures to 50 mL tubes and spin cultures for 10 minutes
6. Remove the supernatant and freeze the pellets. Each bacterial strain had 5 conical tubes for a total of 25 tubes total
7. Combine all pellets into 5mL of lysis buffer (1mL of lysis buffer into each conical tube)
8. Combine all 5 tubes of the same protein into one labeled 15 mL conical tube
9. Incubate on ice for 20 minutes
10. Dounce each 5mL sample on ice 40 times (1 down and 1 up equals 1 time)
11. Clean tissue grinder with water and rinse with lysis buffer between each sample
12. Pellet 4℃ at 10,000 rp, for 15 minutes
13. Prepare the Ni∙NTA Agarose
14. Add 0.5mL Ni∙NTA slurry into a tube. Mix the solution well before pipetting
15. Add 2mL of equilibration buffer per tube to wash
16. Wash 2 times with Equilibration buffer, and drain the buffer between washes
17. Completely drain buffer after the last wash
18. Take the supernatant out of the tubes in the centrifuge and add each to a labeled Ni∙NTA Agarose tube.
19. Rotate the samples at 4℃ for 1 hour-overnight. Remove 100 uL of the input sample and store at -80℃
20. Spin tubes to collect the Ni∙NTA
21. Supernatant sample was taken (100 uL at -80℃)
22. Use 2mL of wash buffer on ice 3 times. Centrifuge between washes for 1 minute at 1,000g to collect supernatant
23. Drain the final wash completely
24. Put 1 mL of elution buffer into the tubes
25. Tip back and forth every minute for 5 minutes on ice
26. Centrifuge all
27. Remove supernatant and collect completely
28. Store all at -80℃.

## SDS Page Gel

### Protocol

1. Add 10 uL of input, 10 uL of supernatant, 2 uL of eluate and 2 uL of agarose to eppendorf tubes. Add 10 uL of buffer to each tube
2. Spin tubes for 1 minute and put in an 80℃ water bath for 10-20 minutes
3. Spin tubes for another minute
4. Load 20 uL of the ladders, input and supernatant into their specific well
5. Load 12 uL of the eluate and agarose to their specific wells
6. Run the gel for 1.5hrs
7. Remove gels, cover with water, and wash 3 times.
8. Add dye to cover the gel for an hour
9. Rinse gel with water
10. Add water to cover the gel and shake overnight
11. Rinse off dye and read gel

## Cloning Protocol

### PCR Clean Up

1. Take 45μl of PCR reaction (ie. Lp , Cf , Dc) and add it to 90μl of NT1 buffer and add both to a column. Place the column in a tube.
2. Spin at max rpm (approximately 1300) for 1 minute.
3. Empty out the tube and add it back to the column. Add 700μl of NT3 buffer.
4. Spin at max rpm for 1 minute.
5. Empty out the tube and add it back to the column. Spin at max rpm for 1 minute.
6. Empty out the tube and dispose of it. Place column in a microfuge tube.
7. Add 40μl of elution buffer. Incubate at room temperature for 1 minute.
8. Spin at max rpm for 1 minute.
9. Dispose of the column.
10. Determine the DNA concentration using a nanodrop device (in ng/μl)
11. Place the microfuge tube containing purified DNA in -20oC storage for later use.

### Gibson's Assembly

1. Each Gibson Assembly mixture is comprised of multiple components. Total volume for each tube in 10μl, with 1μl being vector the volume of DNA and water being dependent on the DNA concentration. Spin all tubes down before any volumes are added.
2. 10μl of 2x Gibson Assembly master max is added to each tube.
3. Incubate each tube for 1 hour at 50oC.

### Transformation

1. Take 2μl of each Gibson Assembly reaction, placing any left over reaction in the -20oC and add it to 50μl of cells (NEB 5 alpha super-competent cells).
2. Place each tube on ice for 30 minutes.
3. Place each tube in a 42oC water bath for exactly 30s.
4. Place each on ice for 5 minutes.
5. Add 950μl of SOC recovery medium to each tube. The total volume of each tube should be approximately 1000μl.
6. Incubate at 37oC for 2 hours.
7. Plate each tube on ampicillin-resistant plates in two volumes, 100μl and 900μl, for a total of 10 plates.
8. Check the plates the next day for colonies.

## Crystal Violet Biofilm Assay

### Materials

* 100 mL M9 media (CONCENTRATION at 1000x) and glutamine (CONCENTRATION at 100x), filtered through a 0.2µm vacuum filtration unit once at 100mL)
* 51 mL LB media (no antibiotics present)
* agar plates containing streaked bacteria of desired strains (DH5α, EMG-2Kλ, NCTC 9001, S. epidermidis, S. aureus)
* 5 50mL conical tubes
* 11 15mL conical tubes
* 4 U-bottom 96 well tissue culture plates
* 2 flat bottom 96 well tissue culture plates
* 0.1% crystal violet (0.2g crystal violet in 200mL water)
* 33.6 mL 30% acetic acid

### Protocol

Note: steps 4-9 should be done within the sterile field of an ethanol-cleaned lab bench under a Bunsen burner to minimize contamination.

1. Add 10 mL LB in 50 mL tubes
2. Pick one colony from each plate (DH5α, EMG-2Kλ, NCTC 9001, S. epidermidis, S. aureus) and put one in each 50 mL tube.
3. Tighten caps then loosen a half turn. Tape the cap on, then incubate in the shaker overnight at 37°C and 175 RPM. CHECK SPECIFICATIONS- Dilute all cultures except NCTC 9001 1:20 (0.5mL culture, 9.5 mL M9) in 15mL conical tubes. Dilute NCTC 9001 1:10 (1mL culture, 9mL M9).
   - Check OD600 of each culture, using a blank of 1:20 LB in M9. (0.25mL LB in 4.75mL M9 is sufficient.)
   - Adjust OD600 to 0.1 using M9 for a total of 10mL, combining diluted culture and M9 into 5 new conical tubes.
   - Check OD600 again to verify it is 0.1.
   - Pipet 100μL of the OD600= 0.1 solution of DH5α into each of the first 6 wells of the first row of each of the four plates. Repeat with the next four strains and the next four rows.
   - Pipet 100μL of M9 into the first 6 wells of the sixth row of each of the four plates.
   - Set two of the plates in the incubator at 37°C. Leave the other two on the lab bench.
   - After 48 hours, take one plate from the incubator and one plate from the bench.
   - Rinse each plate two to three times with distilled water.
   - Pipet 125μL of 0.1% crystal violet into each well previously containing liquid (rows A-F, columns 1-6 of both plates).
   - After 10 minutes, rinse both plates with distilled water three to four times.
   - Pipet 200μL of 30% acetic acid into each well previously containing liquid (rows A-F, columns 1-6 of both plates).
   - Pipet up and down four times then transfer 100mL of each well from the room temperature plate into the corresponding well in a flat bottom 96 well plate. Repeat for the 37°C plate, but transfer into the empty wells (A7 - F12) on the right side of the same flat bottom 96 well plate.
   - Pipet 100μL of 30% acetic acid into each well of row G of the flat bottom 96 well plate.
   - Process OD595 in an absorbance plate reader.
   - After 24 hours, repeat steps 12 - 18 with the remaining plates.

## Coated Protein Trials

### Growing Cells

1. Overnight cultures of S. Epi, S. Aureus, EMG, DH5Alpha, and NCTC were grown in 5 mL of LB (0.5 mL of these were then added into 9.5mL of M9 minimal media and diluted to be between 0.05 and 0.1 (OD600))

### Protein Coated Plates

1. 25 uL of protein, GFP, IA4, IA5, Ze4, and Ze5, were added to each well
2. These dried for 3 hours and any excess protein was removed
3. All plates were put in the incubator for 48 hours at 37 degrees C

### Mixed Protein Plates

1. 100 uL of bacteria were added into each well according to the charts above. On plates 1-4, 10uL of protein were added to specified wells
2. All plates were put in the incubator for 48 hours at 37 degrees C

The crystal violet procedure was followed from this point on

## Gold Particle Preparation for the Gene Gun

[Biolistics PDS-1000/He Particle Delivery System](https://static.igem.org/mediawiki/2018/1/1a/T--WPI_Worcester--biorad_gene_gun_protocol.pdf)

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# Team:WPI Worcester/Results

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# Results

## Biofilm Quantification

![A bar graph of the results of the experiment. Analysis follows this image. D H 5 alpha, E M G 2 K lambda, and N C T C 9001 are all strains of E. coli. They are referred to as DH5α, EMG, and NCTC respectively.]( https://static.igem.org/mediawiki/2018/thumb/0/07/T--WPI_Worcester--maps.jpg/800px-T--WPI_Worcester--maps.jpg.png)

Different strains of E. *coli* and Staphylococcus (S. *aureus*, and S. *epidermidis*) were grown at different temperatures and for different durations to determine ideal incubation conditions for our experiments. DH5α was chosen as a negative control, which it successively showed itself as. As can be seen, of all of the strains, NCTC had the most pronounced growth, with the 72 hr, 37°C condition. However, it has the largest error bars among all strains measured, so its magnitude of growth is suspect. Additionally, with S. *epidermidis* too, overlap in the error bars with the comparable 48 hr, 37°C condition, also questions the statistical significance between growing bacteria at these two conditions. Excluding the negative control, the amount of biofilm produced were inconsistent between strains, which likely means optimal growing conditions depend on the bacterium species in question, given that all are known to produce biofilms. For our trials, 48 hours at 37°C was concluded to be the optimal growing conditions due to the visible distinction between strains while also keeping the time it takes to perform one trial short.

## Antifreeze Proteins against Biofilm Formation

### His-Tag Purification

His-tag purification was conducted to purify antifreeze proteins (IA and Ze) and green fluorescent protein (GFP) from BL21 strains of E. *coli*. These strains produce a larger quantity of proteins. They were activated using IPTG (a mimic of a metabolite of lactose that triggers the lac operon) to produce more protein. The antifreeze proteins and GFP contain histadine, and therefore bind to components of the purification, allowing other parts of the E. *coli* cells to be removed. Two of five clones for both IA and Ze AFPs were used and the proteins purified. For IA, clones 4 and 5 were used (labelled IA4 and IA5) and for Ze, clones 1 and 4 were used (labelled Ze1 and Ze4).

### Protein Concentration Assay

![Graph of Protein Concentration]( https://static.igem.org/mediawiki/2018/6/68/T--WPI_Worcester--COncentrationofProtein.png)

Samples from the eluate of the protein purification were compared with bovine serum albumin (BSA) on a protein assay to quantify the concentration of protein recovered from purification. The graph shows that the purification yielded between 0.42 and 0.54 mg/ml of protein.

### SDS Page Gel

![image of SDS Page Gel]( https://static.igem.org/mediawiki/2018/8/82/T--WPI_Worcester--Gel1.png)
![image of SDS Page Gel]( https://static.igem.org/mediawiki/2018/f/fd/T--WPI_Worcester--GEl2.png)
![image of SDS Page Gel]( https://static.igem.org/mediawiki/2018/5/52/T--WPI_Worcester--Gel3.png)

Purified GFP can be found in well 4 of the first gel. It is around the expected size of GFP, which is 27 kDa. Therefore, it is likely that GFP was purified in this sample. Purified IA4 is seen in well 8 of the first gel, while IA5 can be seen in well 4 of the second gel. Both eluate samples of IA are around 24 kDa, when compared to the ladders in the samples. This is the expected size when compared to literature. Lastly, purified Ze1 can be seen in lane 8 of the second gel and Ze4 can be seen in lane 5 of the third gel. These are the smallest, around 14 kDa, and fall toward the bottom of the gel. Something to note is that there is a band that is present in all gels. It is a non-specific band that seems to not have been purified out using his-tag purification. It is possible that this could contribute to future results of our assays. Future work would require a second round of purification to eliminate this band of proteins before running our assays.

### Biofilm Assay Result

![Graph of Coated Protein Plate Data]( https://static.igem.org/mediawiki/2018/d/da/T--WPI_Worcester--CoatedPlates.png)
![Graph of Mixed Protein Plate Data]( https://static.igem.org/mediawiki/2018/0/0e/T--WPI_Worcester--MixedPlate.png)

The first graph shows the amount of biofilm produced in DH5𝛼, EMG, NCTC, S. *epidermidis*, and S. *aureus* with IA4, IA5, Ze1, Ze4, or GFP coating the well plate. When comparing the red no protein bar to every other bar, most proteins seem to show a reduction in biofilm formation in these trials. This occurs in every strain except for EMG, which shows an increase in biofilm formation. This is likely due to EMG responding quickly to stress, which is what induces biofilm formation to begin with (Kostakioti, 2013).

The second graph shows the amount of biofilm produced in DH5𝛼, EMG, NCTC, S. *epidermidis*, and S. *aureus* with IA4, IA5, Ze1, Ze4, or GFP mixed into the bacteria that was then added to the well plate. When comparing the red no protein bar to every other bar, most proteins seem to show a reduction in biofilm formation in these trials. Similar to the coated plates, this occurs in every strain except for EMG. However, with this method of protein application, the difference is not so dramatic, and the EMG samples with protein mixed in have similar levels of biofilm those which had no protein.

The addition of AFPs or GFP seem to have an impact on the formation of biofilm in all strains that were tested. It is possible that any trends seen in this data could be due to the nonspecific band present during the protein purification protocol.

## Curcumin Assays

![Graph of curcumin assays. Three concentrations of curcumin were used. More detail on the protocol can be found under lab work under protocols.](https://static.igem.org/mediawiki/2018/d/dd/T--WPI_Worcester--CurcuminGraph.jpg)

All five strains, DH5𝛼, EMG, NCTC, S. *epidermidis*, and S. *aureus*, were mixed with the specified amounts of curcumin. As can be seen, the EMG strain had the most pronounced biofilm development. While DH5α had an apparent drop in biofilm development, it like S. epidermidis and S. aureus showed an increase in biofilm growth. NCTC similarly shows an initial drop but an uptick in measured biofilm after 20 µM curcumin solution was applied. However, the overlap between error bars in these four aforementioned strains among ratios means overall measurements may be suspect.

#### Discussion and Future Work

Curcumin’s antibacterial ability against different strains of bacteria was assessed. To see if it had a blanket response among different bacterial species was an objective of this study. Observing the data, particularly the mismatch in drops and increases in biofilm measurement a few reasons for this occurrence arises. The most likely is that there are simply differences in how individual strains of bacteria react to stress. When placed under taxing, harsh conditions nonideal for development, some bacteria form biofilm (Kostakioti, 2013). Curcumin, posited in the literature to kill bacteria, may have proven so harsh for bacteria with membrane deconstructing mechanism that cells went into biofilm development to endure, explaining the increase in biofilm detected in S. *aureus* for example. Another reason may concern the measurement technique used. While great care was taken to be consistent among all assayed plates in all trials, there was no standardization in the initial wash step of the plates to apply the crystal violet stain, which, on those occasions the water was applied for longer than needed, removing the biofilm, thus skewing data.

Future extensions of this project could involve running this experiment once more with the aforementioned issues addressed and remedied. Moreover, discerning a scalable method conducive for industrial production of curcumin from synthetic bacteria (like what is executed currently with insulin) may prove insightful and provide for a case study for a new industry of standardized herbal biologics. If desired, the genes comprising the mechanism of curcumin could be spliced into plant genomes to have the plants produce curcumin themselves.

## Lettuce Colony Counts

![Lettuce colony count graph.](https://static.igem.org/mediawiki/2018/a/a3/T--WPI_Worcester--lettuce_biofilm_graph.png)

As with previous work, five strains of bacteria (DH5𝛼, EMG, NCTC, S. *epidermidis*, and S. *aureus*) were tested for biofilm growth at 37°C for 48 hours. The amount of surviving bacteria were measured instead of the amount of extracellular polymeric substance produced. There are differences seen between this data and that from the crystal violet biofilm assays, most notably the growth of S. *aureus* and the lack of growth of EMG. This further indicates that different strains have different optimal growing conditions. There is also a wide range of standard error, which may be reduced by a lesser dilution before plating on plain LB agar plates and also further standardizing the inoculation of the lettuce leaves.

Further research includes remedying standard error through more even spreading of inoculation media and utilizing a lesser dilution. Ten times what is currently being performed is a recommended starting point (1 to 20,000 instead of 1 to 200,000). Once this is accomplished, trials should be performed with live plants and day and night cycles simulated through temperature and light change.

## Low-Cost Gene Gun Transformation

The low-cost gene gun built by the team was proven to be functional with GFP reporter system. For details on the results, please see the [hardware](https://2018.igem.org/Team%3AWPI_Worcester/Hardware) page.

## Transformation with Antifreeze Proteins

Biofilm assay plates made from transformed cultures of IaAFP, ZeAFP, GFP, Empty psB1C3 plasmid in EMG competent cells. The goal was to induce the various AFPs in bacteria that are known to form biofilms. This was tested on biofilm assay plates containing a sample of each protein with and without arabinose. The results from the biofilm assay plates show that the samples induced with arabinose produced less biofilm when compared to the same protein sample not induced with arabinose. This is because the arabinose binds to the AraC protein and turns on the operon that triggers the expression of the AFPs.

![Graph of Biofilm Assay Trails]( https://static.igem.org/mediawiki/2018/b/bf/T--WPI_Worcester--Bioflim_Assay_Plate.png)

![image of Protein Gel]( https://static.igem.org/mediawiki/2018/7/70/T--WPI_Worcester--ProteinGel1.png)

Image of a protein gel that confirms the expression of the proteins. The protein gel confirms expression of AFPs. The red boxes outline the IaAFP with arabinose, ZeAFP with arabinose and GFP with arabinose all contain bands that show protein expression. The samples of IaAFP, ZeAFP and GFP without arabinose do not show the band that confirms expression of the proteins. The last two wells contained samples of empty psB1C3 plasmid and therefore do not have the bands of protein.

![Image of two microfuge tubes containing G F P cultures, held over U V light and glowing](https://static.igem.org/mediawiki/2018/4/44/T--WPI_Worcester--GFP_cultures.png)

Image of GFP cultures. The two tubes on the right that contain arabinose and the two tubes on the left do not. It can be seen that the tubes containing arabinose are glowing and therefore confirm the expression of the GFPs.

## Project Achievements

We achieved a lot over the course of our project, but encountered many challenges along the way. Because of this, there is a lot of potential for future work.

#### ICEberg Successes

* Purification of AFPs in BL21 strains of E. coli
* Reduction of biofilm formation with any application of His-purified protein
* Transformation of E. coli to express AFPs
* Production of a functional and inexpensive gene gun
* Transformed lettuce leaves to express GFP using the gene gun

#### ICEberg Challenges and Future Directions

* Continue to use difference techniques to attempt to make NCTC-competent cells
* Further purify the AFPs to eliminate the presence of the rouge protein
* Increase the transformation efficiency of GFP from the low-cost gene gun
* Work to standardize the washing step during crystal violet biofilm processing
* Continue to use aseptic technique to reduce the risks of outside contamination

## References

Kostakioti, M., Hadjifrangiskou, M., & Hultgren, S. J. (2013). Bacterial Biofilms: Development, Dispersal, and Therapeutic Strategies in the Dawn of the Postantibiotic Era. Cold Spring Harbor Perspectives in Medicine, 3(4). doi:10.1101/cshperspect.a010306