Experiments

Procedure & Results

Protocol1

Mealworm Dissection

LB Agar Medium Synthesis

Chemical Transformation of E.coli

Materials Needed:

Water(purified water)

LB broth (salt, yeast, 카세린), since we’re making a 500ml solution, we’ll need 12.5g

25g/liter

AGAR (not easily disintegrated by bacteria, structure preserved)

Has to be 1.5% of the solution

Since we’re making a 500ml solution, we’ll need 7.5g

Autoclave

High pressure, high temp sterilization

More than 20 minutes

Procedure:

1. Put water up to 500ml in a graduated cylinder pour it in a large flask

2. Measure LB broth carefully to match 12.5g inside the a plastic temporary container

3. Close the lid to the LB broth, wash the spoon used to transfer it

4. Fold the plastic container in half and pour the LB broth into the flask

5. In the same manner, put AGAR(7.5g) inside the flask

6. Don’t mix he flask, and seal the top of the flask with a tin foil

7. Put it in the autoclave, pour some hot water and close the lid wait for an hour and half

8. When storing the AGAR medium, make sure to preserve it in 60 degree cel.

9. Go to the clean bench, or if it’s unavailable you kindle the alcohol lamp

->(which causes convection and so in a sense forms an umbrella that protects the plate from contamination from the air)

10. Pour the AGAR solution onto eight plates, making sure your hand or your other body part is not hovering over the plate

->(you always have to keep in mind that SOMETHING’s falling from the air.

11. Close the lids of the all eight plates and wait till they harden.

Chemical Transformation

Basic Knowledge:

Competent cell

e-coli’s name : DH5-alpha

DNA to be Added : PUC19

Procedure:

1. Prepare two containers

only the DH5-alpha

DH5 alpha + PUC19 (experimental group)

Put both containers in ice for a minute

2. Raise the temperature abruptly to 42 degrees Cel for a minute

3. Put them into ice for another 2 minutes

4. At the Clean Bench, put LB Medium equally to each container

5. 37°C 180 RPM (at least 30-min)

6. In the centrifuge, 30 seconds at highest speed

7. With a Pipet eliminate remaining precipitate

8. In the LB Medium plate, put appropriate number of glass beads

9. After mixing the substances in the two containers with the pipet,

10. Put the two substances into each separate LB plates, put the lid on top and shake

11. Seal the plate

12. Wait for 2 days until results show up

Chemical Transformation of E.coli

⦁Take competent cells out of -80°C and thaw on ice (approximately 20-30min).

⦁Take agar plates (containing the appropriate antibiotic ) out of 4°C to warm up to room temperature or place in 37°C incubator.

⦁Mix 1μl of DNA (PUC 19) into 50μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.

⦁Place the competent cell/DNA mixture on ice for 20-30min.

⦁Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using).

⦁Put the tubes back on ice for 2 min.

⦁Add 500μl LB without antibiotic) and grow in 37°C shaking incubator for 45min.

Note: This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded on the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.

⦁Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.

Note: We recommend that you plate 50μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

⦁Incubate plates at 37°C overnight.

⦁Place the competent cell/DNA mixture on ice for 20-30min.

⦁Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using).

⦁Put the tubes back on ice for 2 min.

⦁Add 500μl LB without antibiotic) and grow in 37°C shaking incubator for 45min.

Note: This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded on the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.

⦁Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.

Note: We recommend that you plate 50μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

⦁Incubate plates at 37°C overnight.

Result

The basic mechanism of cloning is simple: put gene A into plasmid or the target vector to make it work. With restriction enzyme, we cut the plasmid and put gene A into the part where it is cut out. From this on, the plasmid will be called a backbone. To fit in gene A, we have to make the ends of the cut backbone complementary with gene A. The method of annealing single stranded complementary is by using LIC (Ligation Independent Cloning). We use PCR for this process. Then, we mix gene A and the backbone and insert it in e.coli for transformation. This is how we put PETase into the backbone vector. A bacillus is used to put PETase on the surface to expose it to external surroundings. Our experiment has finished putting PETase and backbone in the e.coli, but we are in the process of verifying our results.

Protocol2 Experiment

1. Confirming that indeed these mealworms digest plastic and recording how much they eat in a day

Experiment1: Influence of light on mealworms

Initial condition
1. Meal worms are in transparent container of its area of base (20X13)cm^2
2. Meal worms' size in average was (2X0.2)cm^2
3. Experiment period : 5/20(Fri)~7/01 (measured every Friday)
4. Meal worms are eating only polystyrene(4.00g)
5. One container is put in shade, another in light
Experiment Result
Date	Shade/Light
		Population [meal worm larva]
		number of imago
		5/20	5/27	6/3	6/10	6/17	6/24	7/1
I-A	light	30	30	30	30	30	30	30
		0	0	0	1	3	3	4
I-B	shade	30	30	30	30	30	30	30
		0	0	0	0	0	0	1

Conclusion

1. Meal worms become imagos faster in light than in shade

Experiment2: How long meal worms can survive only relying on polystyrene

Initial condition
1. Meal worms are in transparent container
2. Meal worms's size in average was (2.6X0.4)cm^2
3. Experiment period : 7/01(Fri)~ (measured once in two weeks)
4. Meal worms are put in shade
5. Meal worms are eating only polystyrene
Experiment Result
Date	7/10	7/15	7/29	8/12	8/26
Meal worm	108	102	92	86	76
Imago(alive+dead)	5	11	19	25	32
Dead meal worm	0	0	2	2	5
Dead Imago	0	0	2	19	32
TOTAL	113	113	113	113	113

Conclusion

1. Imago cannot survive depending only on polystyrene
2. Imagos reproduced, and we could find about 40 larvas that were newly born (there may be more since we couldn't separate the larvas of less than 7mm from excreta)
3. newly born larvas were gathered and grown in another transparent container in sawdust

Experiment3: Decomposition Rate of Polystyrene by Mealworms

Initial condition
1. Group A and B each has 10 meal worms in their container
2. Meal worms in Group A, B are fed only on polystyrene
3. Group C has only polystyrene in its container
4. Group A and C experiment started in 8/24
5. Group B experiment started in 8/25
6. Meal worms are put in shade
Experiment Result
Date	8/24	8/25	8/26	8/27	8/28	8/29	8/30	8/31	9/1	9/2	9/3	9/4	9/5	9/6	9/7	9/8
w_A(t)	4.00	3.99	3.98			3.97	3.97	3.96	3.96	3.96			3.94	3.94	3.93	3.93
w_B(t)			4.00			3.99	3.98	3.97	3.97	3.96			3.96	3.95	3.95	3.94
n_A(t)	10	10	10			9	9	8	8	8			8	8	8	8
n_B(t)			10	10	10	10	10	9	9	9	9	9	8	8	8	8
Date	9/9	9/10	9/11	9/12	9/13	9/14	9/15	9/16	9/17	9/18	9/19	9/20	9/21	9/22	9/23
w_A(t)	3.92			3.91	3.91	3.91	3.91	3.90			3.90	3.90	3.89	3.89	3.89
w_B(t)	3.93			3.92	3.91	3.91	3.91	3.90			3.90	3.90	3.89	3.89	3.89
n_A(t)	7			7	6	6	6	6			6	5	5	5	5
n_B(t)	8			8	7	7	7	7			6	6	6	6	6

After feeding 40 mealworms with only plastic for 5 weeks, we measured the length and the weight of the 20 samples. The average weight is 0.177g per worm and the original length is about 3+- 0.4cm. Compared to the original length 2+-0.2cm, it is noticeable that mealworms are possible to continue their lives with plastics as their only source of nutrient.

Conclusion

1. The average decomposition rate of polystyrene by mealworms was 4.720939905382693E-4g of polystyrene a day.
2. Such decomposition rate can be drew by the formula $\large f(t)=\frac{w(t-1)-w(t)}{n(t)}$ $\large =\frac{1}{t_{f}-t_{i}}\int_{t_{i}}^{t_{f}}f(t)dt$

Team:KoreaSonyeodul/Experiments

Experiments

Procedure & Results

Protocol1

Mealworm Dissection

LB Agar Medium Synthesis

Chemical Transformation of E.coli

Materials Needed:

Water(purified water)

LB broth (salt, yeast, 카세린), since we’re making a 500ml solution, we’ll need 12.5g

25g/liter

AGAR (not easily disintegrated by bacteria, structure preserved)

Has to be 1.5% of the solution

Since we’re making a 500ml solution, we’ll need 7.5g

Autoclave

High pressure, high temp sterilization

More than 20 minutes

Procedure:

1. Put water up to 500ml in a graduated cylinder pour it in a large flask

2. Measure LB broth carefully to match 12.5g inside the a plastic temporary container

3. Close the lid to the LB broth, wash the spoon used to transfer it

4. Fold the plastic container in half and pour the LB broth into the flask

5. In the same manner, put AGAR(7.5g) inside the flask

6. Don’t mix he flask, and seal the top of the flask with a tin foil

7. Put it in the autoclave, pour some hot water and close the lid wait for an hour and half

8. When storing the AGAR medium, make sure to preserve it in 60 degree cel.

9. Go to the clean bench, or if it’s unavailable you kindle the alcohol lamp

->(which causes convection and so in a sense forms an umbrella that protects the plate from contamination from the air)

10. Pour the AGAR solution onto eight plates, making sure your hand or your other body part is not hovering over the plate

->(you always have to keep in mind that SOMETHING’s falling from the air.

11. Close the lids of the all eight plates and wait till they harden.

Chemical Transformation

Basic Knowledge:

Competent cell

e-coli’s name : DH5-alpha

DNA to be Added : PUC19

Procedure:

1. Prepare two containers

only the DH5-alpha

DH5 alpha + PUC19 (experimental group)

Put both containers in ice for a minute

2. Raise the temperature abruptly to 42 degrees Cel for a minute

3. Put them into ice for another 2 minutes

4. At the Clean Bench, put LB Medium equally to each container

5. 37°C 180 RPM (at least 30-min)

6. In the centrifuge, 30 seconds at highest speed

7. With a Pipet eliminate remaining precipitate

8. In the LB Medium plate, put appropriate number of glass beads

9. After mixing the substances in the two containers with the pipet,

10. Put the two substances into each separate LB plates, put the lid on top and shake

11. Seal the plate

12. Wait for 2 days until results show up

Chemical Transformation of E.coli

Chemical Transformation of E.coli

⦁Take competent cells out of -80°C and thaw on ice (approximately 20-30min).

⦁Take agar plates (containing the appropriate antibiotic ) out of 4°C to warm up to room temperature or place in 37°C incubator.

⦁Mix 1μl of DNA (PUC 19) into 50μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.

⦁Place the competent cell/DNA mixture on ice for 20-30min.

⦁Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using).

⦁Put the tubes back on ice for 2 min.

⦁Add 500μl LB without antibiotic) and grow in 37°C shaking incubator for 45min.

⦁Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.

Note: We recommend that you plate 50μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

⦁Incubate plates at 37°C overnight.

⦁Place the competent cell/DNA mixture on ice for 20-30min.

⦁Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using).

⦁Put the tubes back on ice for 2 min.

⦁Add 500μl LB without antibiotic) and grow in 37°C shaking incubator for 45min.

⦁Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.

Note: We recommend that you plate 50μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

⦁Incubate plates at 37°C overnight.

Result

Protocol2 Experiment

1. Confirming that indeed these mealworms digest plastic and recording how much they eat in a day