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Algae Genetic Modification

Co-Culture Lab

Names of each student: Salem Mutata, Leyao Wang, Haozhe Yao, Jingyi Xia, Rui Gong

Subteam name: Co-Culture team

July

Date: July 11, 2024

Protocols Performed: Put 2 500mL culture bottles in autoclave for sterilization. Add 200mL TAP medium into each bottle. Labeled them as bottle A and bottle B. Pick 1cm size medium (solid) with algae and add to bottles separately. Put the culture bottles in incubator (in 314), at 26 ℃, 110 rpm, LED light, for overnight culture.

Purpose: Autoclave Build the experimental setup Start culturing algae

Supplies used/Materials: 2 500mL culture bottles, TAP medium, algae

Results:

Conclusions/Next Lab goals: Next time we will get the OD reading to see if the algae grow.

Extra notes:

Names of each student: Salem Mutata, Leyao Wang, Haozhe Yao, Jingyi Xia, Rui Gong

Subteam name: Co-Culture team

Date: July 12, 2024

Protocols Performed: Clean the cuvette with 70%ethanol. Preheat the spectrophotometer, set the wavelength to 750mm. Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use pipette gun to get 1mL algae solution from bottle A and bottle B separately. Put them into spectrometer to get OD reading.

Purpose: Get OD reading

Supplies used/Materials: Ethanol, cuvette, alage solution

Results: A: 0.006A B: 0.005A

Conclusions/Next Lab goals: There is no significant change in culture bottles. We will wait for tomorrow’s result to determine whether we should start a new culture.

Extra notes:

Names of each student: Salem Mutata, Leyao Wang, Haozhe Yao, Jingyi Xia, Rui Gong

Subteam name: Co-Culture team

Date: July 13, 2024

Protocols Performed: Get OD reading: Set a blank using 1mL TAP medium. Set samples using 1mL liquid from bottle A and bottle B separately. Put the culture bottle to the shaker (in NSC 112) at indoor temperature, 110rpm, LED light. Use pipette gun to get 10mL Growth medium. Put it in 2 new EP tubes separately, label them as 1,2. Add 1cm x 1cm solid medium with algae. Put 10 mL TAP medium in another EP tube, label it as 0. Culture them in the same incubator.

Purpose: Get OD reading, transform culture bottles to another shaker, make a new culture with Growth medium.

Supplies used/Materials: TAP medium, Growth medium, EP tube, algae

Results: A: 0.020A B: 0.012A

Conclusions/Next Lab goals:

Extra notes:

Names of each student: Salem Mutata, Leyao Wang, Haozhe Yao, Jingyi Xia, Rui Gong

Subteam name: Co-Culture team

Date: July 15, 2024

Protocols Performed: 1. Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. 2. Set samples: use pipette gun to get 1mL algae solution from bottle A and bottle B separately. Put them into spectrometer to get OD reading.

Purpose: Get OD reading

Supplies used/Materials: Ethanol, cuvette, alage solution

Results: A: 0.224A B: 0.112A

Conclusions/Next Lab goals: The algae solution turn green obviously.

Extra notes:

Date: July 16, 2024

Add 1 ml microalgae from group A/B into each EP tube. (5 in total) Add 9ml TAP into each EP tube. Add ethanol to each EP tube. ( The volume concentration of ethanol in the solution is 0% (group 0), 0.8% (group 1), 1.1% (group 2), 1.4% (group 3), 1.7% (group 4)

  Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Notebook Page

Purpose: Explore the influence of the ethanol to the growth of the monoculture microalgae

Supplies used/Materials: Ethanol, microalgae, TAP medium.

Results: Group A: 0.552 Group B: 0.662 Conclusions/Next Lab goals:

Extra notes: Date: July 17, 2024

Protocols Performed: Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use pipette gun to get 1mL algae solution from bottle A and bottle B and ethanol resistant group separately. Put them into spectrometer to get OD reading.

   Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Making a growth curve and exploring the influence of ethanol to the growth of the monoculture.

Supplies used/Materials:

Results: Group 0: 0.085 Group 1:0.107 Group 2: 0.160 Group 3: 0.122 Group 4: 0.099 Group A: 0.888 Group B: 0.945

Conclusions/Next Lab goals:

Extra notes:

Date: July 18, 2024

Culturing E.coli: E.coli is transferred into a small EP tube (1ml), shaking in condition 220 RPM, 37 degree celsius for 2 hours Add 200 ml LB media and 1 ml culture above into a flask. And incubate it in condition 100 RPM and, 37 degree celsius for one night.

   Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Making a growth curve and exploring the influence of ethanol to the growth of the monoculture.

Supplies used/Materials:

Results: Group 0: 0.295 Group 1: 0.300 Group 2: 0.544 Group 3: 0.270 Group 4: 0.195 Group A: 0.721 Group B: 0.758

Conclusions/Next Lab goals:

Extra notes:

Date: July 19, 2024

Set a blank: use pipette gun to get 1mL LB medium in the cuvette. Press “set ref”. Set samples: use pipette gun to get 1mL algae solution from bottle E.coli culture. Put them into spectrometer to get OD reading. (OD 600)

Co-Culture protocol

Co-culture of E. coli with microalgae: Inoculating microalgae and E.coli with ratio 1:1 2:1 and 9:1 in three separate flasks (microalgae: E. coli) ( 8 ml E.coli, 8 ml, 16 ml, 72 ml microalgae) ( In this experiment, the microalgae used has OD 750 = 0.127, E. coli has OD 600 =1.567 Add TAP medium to the co-culture until the total volume reaches 150 ml in each flask.

Add 50 ml fresh TAP medium into group A monoculture microalgae for the comparison with group monoculture microalgae.

Measuring biomass Protocol

Measuring biomass: The samples used in measuring OD values are not discarded. Instead, they are transferred into a centrifuge tube separately. (The weight of each empty centrifuge tube is measured before). Centrifuging those tubes in the condition 10000 RPM, 10 min. Discard the supernatant, and put the tube into the drier. Measure the weight of the centrifuge tube with the dried pellet.

   Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Making an E.coli growth curve and monoculture microalgae growth curve. Exploring the influence of the ethanol

Supplies used/Materials: TAP medium, LB medium. Results: Group 0: 0.373 Group 1: 0.458 Group 2: 0.573 Group 3: 0.459 Group 4: 0.340 Group A: 0.634 Group B: 0.803

Conclusions/Next Lab goals:

Extra notes:

Date: July 20, 2024

    Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Making an E.coli growth curve and monoculture microalgae growth curve. Exploring the influence of the ethanol

Supplies used/Materials:

Results: Group 0: 0.251 Group 1: 0.646 Group 2: 0.820 Group 3: 0.645 Group 4: 0.496 Group A: 0.634 Group B: 0.803

In the figure above, Day 1 is today (July 20, 2024)

In the figure above, 96.00h is today.

Conclusions/Next Lab goals:

Extra notes:

Date: July 21, 2024

    Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose:

Making an E.coli growth curve and monoculture microalgae growth curve. Exploring the influence of the ethanol

Supplies used/Materials:

Results: Shown in the figures in July 20 th.

Conclusions/Next Lab goals:

Extra notes:

Date: July 22, 2024

PHB extraction: Transferred 75ml microalgae in total from microalgae monoculture group B into 2 EP tubes. Centrifuge the EP tube with 5000 rpm for 10 min, and then discard the supernatant. Put the EP tubes into the drier. Add 2.5 ml sodium hypochlorite to the biomass in each tube and incubate for 2h in a water bath at 30 degrees celsius. After incubation, centrifuge EP tubes at 5000 rpm for 15 minutes. The pellet was washed with 0.25ml deionized water and 0.25ml of a solution of acetone and ethyl alcohol for each EP tube. The pellet was dissolved by adding 5 ml of warm chloroform to extract PHB.

    Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose:

Supplies used/Materials: Sodium hypochlorite, acetone, ethyl alcohol and chloroform. TAP media, LB media, cuvette, scale, centrifuge tube …… Results: See the figures on July 20th.

Conclusions/Next Lab goals:

Extra notes:

Date: July 23, 2024

   Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Making an E.coli growth curve, monoculture microalgae growth curve and co-culture growth curve. Exploring the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: TAP media, LB media, cuvette, scale, centrifuge tube ……

Results: Biomass data, see figures in July 20th Group 0: 0.911 Group 1: 1.484 Group 2: 1.359 Group 3: 1.340 Group 4: 1.338 Group A: 0.958 Group B: 0.971

Conclusions/Next Lab goals:

Extra notes:

Date: July 24, 2024

Cryopreservation of microalgae: 1. Add 1 ml microalgae, 4ml 15% glycerol into a tube and seal the joint between the cap and the body of the bottle with a sealing film. Preserve the tube in -80 degrees celsius. (Method 1)

          (Method 2)
          1. Dispense solution (5% DMSO, 5% EG, 5% proline, v/v)
          2. Add equal amounts of microalgae and the solution to a tube and seal it. 
           Put the tube on an ice-bath for over 15 minutes and then put it in -40 degrees freezers
           for 4 hours   
          3. Transferred it into -80 degrees freezer for storage.

Cryopreservation of E. coli 1. Add 500 ul of the bacteria culture to 500 ul of 50% glycerol in a 2 ml screw top tube and gently mix. 2, Freeze the glycerol stock tube at -80 degrees celsius.

   Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Preserve the sample for future use.

Supplies used/Materials: Glycerol, DMSO, EG, proline, LB media, TAP media……

Results: Nobiomass data Co-culture OD, see figures on July 20th. (Day 5) OD 750 Group 0: 0.940 Group 1: 1.515 Group 2: 1.392 Group 3: 1.332 Group 4: 1.376 Group A: 0.967 Group B: 0.988

Conclusions/Next Lab goals:

Extra notes:

Date: July 25, 2024

Protocols Performed: Preservation of the samples: Transfer all of the culture into EP tubes and centrifuge them for 5000 rpm, 10 min. Discard the supernatant and put the tube into the drier. Put those tubes into the -80 degrees celsius freezer.

Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use pipette gun to get 1mL algae solution from ethanol resistant group separately. Put them into spectrometer to get OD reading.

    Please refer to the paper lab note for other experiments and details, ( 2024 Group 4 Haozhe Yao Eric) Lab notebook.
  If the above information is different from the paper lab notes, the paper lab notes shall prevail.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: EP tubes, TAP media….

Results: OD 750 Group 0: 0.940 Group 1: 1.571 Group 2: 1.402 Group 3: 1.356 Group 4: 1.364

Conclusions/Next Lab goals:

Extra notes:

August 2024

Names of each student: CJ Ume…

Date: August 1, 2024

Purpose: LB/agar plate with ampicillin LB/agar plates without antibiotics LB/agar with chloramphenicol

-Make sure this is done next to a bunsen burner so the LB remains sterilized

Protocols Performed: LB with ampicillin Combine 20g of LB Agar and 500ml of Distilled water into a 1L container. Autoclave for 30 minutes (liquid) - use autoclave foil to cover the top. Let it cool to 55°C (use thermometer to check temperature) Add 0.125g of Ampicillin (swirl just a little so it doesn’t solidify) Before continuing, make sure step 6 is done next to a bunsen burner Carefully pour the mixture onto the plates, only filling it halfway. Let the gel solidify in the plates, then flip them upside down, stack them top of eachother, use tape to hold them, then place in the fridge.

Protocols Performed: LB w/o antibiotics Combine 20g of LB Agar and 500ml of Distilled water into a 1L container. Autoclave for 30 minutes (liquid) - use autoclave foil to cover the top. Let it cool to 55°C (use thermometer to check temperature) Swirl a little so it doesn’t solidify Before continuing, make sure step 6 is done next to a bunsen burner Carefully pour the mixture onto the plates, only filling it halfway. Let the gel solidify in the plates, then flip them upside down, stack them on top of eachother, use tape to hold them, then place in the fridge.

Protocols Performed: LB with chloramphenicol Just take 250ml of the prepared LB without antibiotics once it has cooled to 55°C Add 250 μl of chloramphenicol Before continuing, make sure step 6 is done next to a bunsen burner Carefully pour the mixture onto the plates, only filling it halfway. Let the gel solidify in the plates, then flip them upside down, stack them on top of eachother, use tape to hold them, then place in the fridge.

Supplies used/Materials: LB Agar, Distilled water, Ampicillin, chloramphenicol, 1L container, bunsen burner

Results:

Date: August 6th

Protocols Performed:

Add 200mL TAP medium into one bottle. Pick 1cm size medium (solid) with algae and Put the culture bottles in incubator (in nsc 110), at 26 ℃, 110 rpm, LED light, for overnight culture.

Purpose: Prepare for future experiments and prepare for the growth of monoculture microalgae.

Supplies used/Materials: Glass bottle, TAP media….

Results: Conclusions/Next Lab goals: The next lab goals is to Take OD readings of algae biomass.

Extra notes: The 1 cm solid was mistaken for the algae glycerol stock but it turned out it was the algae biomass. It is not expected for the algae biomass to have much growth, but we decided to keep it to assess its growth in TAP medium.

Date: August 7th

Protocols Performed: Add 1 cm solid of algae to 200 ml TAP medium in a glass container. Let the solution shake in a shaking incubator shaking at 110 rpm and at 26 degrees celsius overnight. An LED light was placed in the shaking incubator to also stimulate growth.

Purpose: Prepare for future experiments, explore what conditions of an incubator influence growth of a monoculture algae

Supplies used/Materials: TAP media, shaking incubator, micropipette

Results: To be determined

Conclusions/Next Lab goals:

Extra notes: It was discovered the 1 cm of solid algae that was accidentally mistaken as algae glycerol stock was discovered to be algae biomass.

SubTeam member names: Chloe Benjamin and Sebastian Haines

Subteam name: Co-culture team

Date: August 8th

Protocols Performed: Measuring algae biomass Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from ethanol resistant group separately. Put them into a spectrometer to get OD reading. Record OD reading Preparing algae glycerol stock and tubes 1-5 (containing algae + ethanol) Autoclave an erlenmeyer flask that will be used for the algae glycerol stock to grow in. Let the algae glycerol stock and thaw and then add it to 200 Add the following volumes of 100 percent ethanol to achieve the desired ethanol concentrations in the remaining 19 mL.

Tube 1-For 0.8% ethanol: 0.152 mL Tube 2-For 1.1% ethanol: 0.209 mL Tube 3-For 1.4% ethanol: 0.266 mL Tube 4-For 1.7% ethanol: 0.323 mL Tube 5-For 2.0% ethanol: 0.38 mL After adding the ethanol, top up with the tap medium to reach a total volume of 19 mL, and then add the 1 mL of algae stock to reach the final volume of 20 mL in each of the five falcon tubes. Place the algae glycerol stock and the falcon tubes containing both ethanol and algae in the shaker to shake at 110 rpm and at 26 degrees celsius.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae as well as the growth of algae previously preserved in glycerol
Autoclave Build the experimental setup and Start culturing algae in other conditions

Supplies used/Materials: Falcon tubes, TAP media, algae glycerol, ethanol, pipette gun, erlenmeyer flask

Results: OD 750 Algae biomass: 0.187

Conclusions/Next Lab goals: Take OD readings of the algae glycerol stock and the algae mixed with ethanol.

Extra notes:

Subteam members: Sebastian Haines and Will Switzer

Subteam name: Co-culture team

Date: August 9th

Protocols Performed:

Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Record OD readings. Place all samples back into the incubator to shake at 110 rpm and at 26 degrees celsius overnight. An LED light was placed inside the incubator to further stimulate algae growth.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvette, and micropipette

Results: OD 750 Algae biomass: 0.514A Algae glycerol: 0.383 A Tube 1: 0.618A Tube 2: 0.959A Tube 3: 1.236A Tube 4: 0.018A Tube 5: 0.563A

Conclusions/Next Lab goals: Record OD readings.

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture team

Date: August 10th

Protocols Performed:

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvette, and micropipette

Results: Algae biomass: 0.302 A Algae glycerol: 0.938A Tube 1: 0.257 A Tube 2: 0.150A Tube 3: 0.133 A Tube 4: 0.155A Tube 5: 0.153 A

Conclusion:

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture team

Date: August 11th

Protocols Performed:

Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Record OD readings. Place all samples back into the incubator to shake at 110 rpm at 26 degrees celsius overnight. An LED light was placed inside the incubator to further stimulate algae growth.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvette, and micropipette

Results: Algae biomass: 0.278A Algae glycerol: 1.233 A Tube 1: 0.417 A Tube 2: 0.288 A Tube 3: 0.250A Tube 4: 0.295 A Tube 5: 0.215 A

Conclusion/Next lab goals: Record OD readings.

Extra notes:

Subteam members: Sebastian Haines

Subteam name: Co-culture team

Date: August 12th

Protocols Performed: Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Record OD readings. Place all samples back into the incubator to shake at 110 rpm at 26 degrees celsius.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvette, and micropipette

Results: OD 750 Algae biomass: 0.212 A time stamp: 3:43 pm Algae glycerol stock: 1.187 A time stamp: 3:53 pm Tube 1: 0.313 A time stamp: 4:01 pm Tube 2: 0.366 A time stamp: 4:14 pm Tube 3: 0.204 A time stamp: 4:19 pm Tube 4: 0.195 A time stamp: 4:24 pm Tube 5: 0.122 A time stamp: 4:31 pm

Conclusions/Next Lab goals: The next step is continuing to record OD readings.

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture

Date: August 13th

Protocols Performed: Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Place all samples back into the incubator to shake at 110 rpm at 26 degrees celsius overnight. An LED light was placed inside the incubator to further stimulate algae growth.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: spectrometer, cuvette, and micropipette

Results: OD 750 Algae biomass: 0.213 A
Algae glycerol stock: 1.088 A Tube 1: 0.451 A Tube 2: 0.504 A Tube 3: 0.529 A Tube 4: 0.214 A Tube 5: 0.298 A

Conclusions/Next Lab goals: The next step is continuing to record OD readings.

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture Team

Date: August 14th

Protocols Performed:

Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Place all samples back into the incubator to shake at 110 rpm at 26 degrees celsius.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvettes…

Results: OD 750 Algae biomass: 0.126 A Algae glycerol stock: 0.947 A Tube 1: 0.744 A Tube 2: 0.719 A Tube 3: 0.557 A Tube 4: 0.553 A Tube 5: 0.269 A

Conclusions/Next Lab goals: The next step is continuing to record OD readings.

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture Team

Date: August 15th

Protocols Performed Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Place all samples back into the incubator to shake at 110 rpm and at 26 degrees celsius overnight. An LED light was placed inside the incubator to further stimulate algae growth.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer and cuvettes

Results: OD 750 Algae biomass: 0.155 A Algae glycerol stock: 0.814 A Tube 1: 0.774 A Tube 2: 1.129 A Tube 3: 1.118 A Tube 4: 0.980 A Tube 5: 0.519 A

Conclusions/Next Lab goals: The next step is continuing to record OD readings.

Extra notes:

Subteam members: Chloe Benjamin and Amy Nguyen

Subteam name: Co-culture Team

Date: August 16th

Protocols Performed: Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Place all samples back into the incubator to shake at 110 rpm at 26 degrees celsius.

Purpose: Prepare for future experiments, explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvettes, and micropipette

Results: OD 750 Algae biomass: 0.175 A Algae glycerol stock: 0.783 Tube 1: 1.047 Tube 2: 1.299 A Tube 3: 1.003 A Tube 4: 1.179 A Tube 5: 0.980 A

Conclusions/Next Lab goals: The next step is continuing to record OD readings.

Extra notes:

Subteam members: Chloe Benjamin

Subteam name: Co-culture Team

Date: August 26th

Protocols Performed: Set a blank: use a micropipette to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use a micropipette to get 1mL algae solution from each group separately. Put them into a spectrometer to get OD reading.
Place all samples back into the incubator to shake at 110 rpm and at 26 degrees celsius overnight. An LED light was placed inside the incubator to further stimulate algae growth.

Purpose: Explore the influence of ethanol to the growth of monoculture microalgae.

Supplies used/Materials: Spectrometer, cuvettes, and micropipette

Results: OD 750 Algae glycerol stock: 0.504 A

Conclusions/Next Lab goals: As seen on the growth curves on the next page, it is indicated growth occurred in the algae glycerol stock and the algae with ethanol added but growth did decrease between certain dates in these samples as well. As indicated by the second graph on the next page, there was evidence that increasing ethanol concentrations resulted in increasing algae growth at certain concentrations of ethanol and at certain dates. The ethanol concentration of 1.1% was correlated with the highest algae growth by the last date of the reading. There were unknown substances seemingly growing in the certain falcon tubes, which is evidence of contamination. It was concluded the next best step is to restart the experiments to regrow an algae monoculture and assess the influence of ethanol concentrations on algae growth.

Extra notes: There was no remaining liquid to take an OD reading of in tubes 1-5.

                Algae Biomass Growth

August 8th August 9th August 10th August 11th August 12th August 13th August 14th August 15th August 16th Algae biomass 0.187 A 0.514 A 0.302 A 0.278 A 0.212 A 0.213 A 0.126 A 0.155 A 0.175 A

September

Date: September 9, 2024

Protocols Performed: Put 2 500mL, 5 250 ml culture bottles in autoclave for sterilization. Prepped incubator, hanged light

Purpose: Autoclave Build the experimental setup

Supplies used/Materials: culture bottles, autoclave, incubator, light

Results:

Conclusions/Next Lab goals: Start an Algae culture and Ethanol Experiment with different values

Extra notes:

Date: September 10, 2024

Protocols Performed: Tap medium was cooled to room temperature Add 100 ml of TAP medium into 500 ml culture bottle lab and label “algae” Thaw algae cells using a water bath at 35 degrees for 3 minutes while swirling it. ETOH and Tap mediums solutions were created in 250 ml bottles to make concentrations 20 %, 33 %, 50 %, 75% and 90% etoh and were labeled according to concentration. Pick 1cm size medium (solid) with algae and add to bottles separately. Put the culture bottles in an incubator at 26 ℃, 110 rpm, LED light, for overnight culture.

An example of one concentration is 50% ethanol Ethanol: 15 mL TAP medium: 15 mL Total: 30 mL

Purpose: Start algae culture Start Ethanol experiment to determine the effect of etoh on algae using more etoh values.

Supplies used/Materials: culture bottles, algae, tap medium, etoh, water bath, incubator, light

Results:

Conclusions/Next Lab goals: Monitor and get OD readings

Extra notes:

Date: September 11, 2024

Protocols Performed:

Clean the cuvette with 70%ethanol. Start the spectrophotometer, set the wavelength to 750mm. Set a blank: use pipette gun to get 1mL TAP medium in the cuvette. Press “set ref”. Set samples: use pipette gun to get 1mL of samples. Put them into spectrometer to get OD reading.

Purpose: Get OD readings

Supplies used/Materials: etoh,cuvette, sample cultures, tap medium

Results:

Algae 20% etoh 32% etoh 50% etoh 75% etoh 90% etoh 0.221A 1:158A 1.024A 0.950A 0.785A 0.570A

Conclusions/Next Lab goals: Continue to Monitor OD readings Practice PHB extractions

Extra notes: Refer to Yao Haozhe for PHB related notes

Date: September 12, 2024

Protocols Performed:

Purpose: Get OD readings

Supplies used/Materials: etoh,cuvette, sample cultures, tap medium

Results:

Algae 20% etoh 32% etoh 50% etoh 75% etoh 90% etoh 0.386A 0.385 0.269 -0.006 -0.011 0.125

Conclusions/Next Lab goals: Continue to Monitor OD readings

Extra notes:

Date: September 13, 2024

Protocols Performed:

Purpose: Get OD readings

Supplies used/Materials: etoh,cuvette, sample cultures, tap medium

Results:

Algae 20% etoh 32% etoh 50% etoh 75% etoh 90% etoh 0.360A 0.0012 -0.035 -0.005 -0.503 -0.0207

Conclusions/Next Lab goals: Continue to Monitor OD readings

Extra notes: Results went down in value so much, confirm again tomorrow.

Date: September 14, 2024

Protocols Performed:

Autoclave 2 250 ml bottles Culture a new batch of algae using the same procedure as Sept 10.

Purpose: Prep for new algae culture

Supplies used/Materials: Refer to Sept 10

Results:

Conclusions/Next Lab goals: Get OD readings next day

Extra notes: After observing the existing algae batch, we suspect it was contaminated, so a new batch was prepared. The algae had also turned a whitish/greyish color instead of the usual green. We stopped adding ethanol because we believe the algae couldn’t recover in such a high concentration. The new plan is to take samples from a subset of the fresh culture rather than directly from stock. The new batch was made using algae stocks from the summer.

Date: September 15, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.217A

Conclusions/Next Lab goals: Continue to monitor OD readings

Extra notes:

Date: September 16, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.292A

Conclusions/Next Lab goals: Continue to monitor OD readings

Extra notes:

Date: September 17, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.226 A

Conclusions/Next Lab goals: Continue to monitor OD readings

Extra notes: The OD readings have dropped again, and the algae are looking pale and whitish once more. We will observe for one more day, but we may need to restart the culture if there’s no improvement.

Date: September 18, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.173 A

Conclusions/Next Lab goals: Start a new culture of algae

Extra notes: This culture is a bust again. Restart algae experiment again.

Date: September 19, 2024 Protocols Performed:

Culture a new batch of algae using the same procedure as Sept 10.

Purpose: Prep for new algae culture

Supplies used/Materials: Refer to Sept 10

Results:

Conclusions/Next Lab goals: Get OD readings next day

Date: September 20, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.243A

Conclusions/Next Lab goals: Continue to monitor OD readings

Date: September 21, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.341A

Conclusions/Next Lab goals: Continue to monitor OD readings

Extra notes:

Date: September 22, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.217A

Conclusions/Next Lab goals: Continue to monitor OD readings

Extra Comments: The algae culture appears to have failed again. After consulting with Dr. Sylvester, we suspect it’s contaminated with bacteria. We’ve ordered new algae stock and TAP medium and are waiting for their delivery. In the meantime, we’ll look for an alternative PHB extraction protocol.

Date: September 23, 2024

Protocols Performed: Refer to Sept 11 Purpose: OD readings of new algae culture

Supplies used/Materials: Refer to September 11

Results: 0.185 A

Conclusions/Next Lab goals: Clean algae room and disinfect

Extra comments: The OD went down again. We will clean the room and try again with new stocks.

PHB

Test (PHB extraction from E. coli) We search for many studies involve PHB production and characterization, the simplest way of characterizing its strong absorption in UV region. (Around 240-250 nm) Here is the brief description of the step we use to extract PHB 1. 75 ml of culture was centrifuged at 5000rpm for 15 min. The supernatant was discarded and the pellet was reserved. 2. The pellet was treated with 10 ml sodium hypochlorite and the mixture was incubated at 30 degrees Celsius. 3. The mixture was centrifuged at 5000rpm for 15 min and then washed with distilled water, acetone and methanol respectively. 4. The pellet was dissolved in 5ml boiling chloroform and evaporates by pouring the solution on glass tray. 5. When measuring, the PHB is dissolved in chloroform again and transferred into quartz cuvette for UV spectrophotometer analysis using pure chloroform as blank.

Here is the graph we receive

There exists a peak at 249nm, which shows our extraction is successful. The sample will also be sent for FTIR spectrophotometer analysis of PHB.

We quantify PHB concentration by using sulfuric acid digestion method 1. The PHB we extract is add 10ml concentrated sulfuric acid and the solution was heated for 10min at 100 degrees Celsius. In this process, PHB is converted to crotonic acid 2. After the solution cooled, the reaction product is transferred into a quartz cuvette for analysis at 235nm using concentrated sulfuric acid as blank. 3. After that, we apply standard curve of crotonic acid to this OD reading to calculate the concentration of the PHB.