Goal:

1. Meeting with Dr. Chin
2. Transformations for acs and CreiLOV constructs
3. Restriction digest with acs and CreiLOV constructs

Meeting:

1. With Dr. Kuk-Jeong Chin (anaerobic bacteria specialist at GSU)
2. Said that our project idea was novel and had potential, but that it was better suited for a phD student
3. Anaerobic bacteria have extremely long doubling times of weeks to months
4. Requires anaerobic facility to maintain constant flow of gasses
   • Even using Hungate technique would require near constant maintenance and exchange of gasses
5. Every anaerobic bacteria has very different requirements, but they are all very fragile
   • Need to be babied
   • Anaerobic conditions are hard to maintain
6. Suggested that we look into a facultative anaerobe instead of an obligate anaerobe
   • Suggested something with nitrogen-fixation, ammonia, or some other toxin that could be turned into something useful

Transformation:

1. Dilute DNA sample from IDT with 50 microliters TF buffer.
2. Dilute DNA with 1/100 of DNA and water.
3. Combine 1 tube of electrically competent DH5a cells and 1 μL of DNA dilution (acs and CreiLOV respectively)
4. Transfer the contents of the Eppendorf tube to a cuvette and tap the cuvette on the table to evenly distribute the contents and to get rid of air bubbles.
5. Place the cuvette into the Bio-Rad MicroPulser and deliver the electric shock.
6. Immediately after, add 900 μL SOC medium to the cuvette and micropipette mixed the solution.
7. Transfer the solution from the cuvette to a shaker tube and place in the shaker at 37°C at 200 rpm for 1 hour.
8. After shaking for 1 hour, streak 150 μL of the solution onto an agar plate with the respective antibiotics.
9. Incubate plates at 37°C for at least 24 hours.

Restriction Digest:

1. Prepare a 3x Fast Digest concentration cocktail with the following proportions: 1 μL EcoRI, 1 μL PstI, 3 μL of 10X Fast Digest Buffer, and 12.5 μL of diH2O.
2. Add 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then add 12.5 μL of DNA
3. Incubate at 37° C for 30 minutes.
4. Place in freezer to preserve for later gel extraction.

Results:

Goal:

1. Meet with Cécilia from the Toulouse iGEM team
2. Midi-prep on overnight cultures
3. Make glycerol stocks from overnight cultures

Meeting:

1. With Cécilia (student leader of Toulouse iGEM team)
2. Worked very closely with a phD student that had experience with anaerobic bacteria, although not the exact strain they used
3. Talked to specialist of their bacteria C. ljungdahlii
   • Highly recommended that we work with a specialist on E. limosum
4. Had challenges finding a good medium
   • Rich medium was unsuccessful
   • Used a minimal media
5. Na2S was their reducing agent
   • Had to be refrigerated and desiccated
   • Kept away from water or else it would lose reducing power
6. Said that doubling time for C. ljungdahlii was between 2 days and a week
   • Much less than Dr. Chin suggested
7. Was able to get to OD of 0.3 in 50 hours
   • Achieved exponential growth phase
8. Did not attempt semi-aerobic co-culture
   • Had major safety problems with combining hydrogen and oxygen gas
   • Hydrogen gas, even on its own, had to be below 4% concentration, or else it would explode
   • Suggested a two-stage culture so that hydrogen and oxygen could be kept separate
9. Very difficult to work with lyophilized (freeze-dried) sample
   • Recommended we purchase live bacteria even though it is more expensive
10. Used a device to split water into H2 and O2 to maintain low concentration of both gasses
    • Not sure what she meant by this
    • Works backwards from what I thought she said?

Goal:

1. Midi-prep of ACS and CreiLoV

Protocol:

Purpose: Extract plasmid DNA (larger quantity)

QIAGEN Plasmid Midi Kit Protocol

1. Separated 100 mL of bacterial overnight culture into 5 separate 50 mL falcon tubes and centrifuged at 6,000 rpm for 15 minutes at 4°C.
2. Decanted supernatant.
3. Added 4 mL of Buffer P1 to one tube, pipet mixed, and transferred to another tube. Mixed and transferred contents to the next tube with pelleted cells. Repeated until all tubes were combined.
4. Added 4 mL of Buffer P2 to the tube containing 4 mL of Buffer P1 and the combined resuspended pelleted cells and vigorously inverted 6 times.
5. Incubated at room temperature for 3 minutes.
6. Added 4 mL of Buffer P3 and vigorously inverted 10 times.
7. Incubated on ice for 15 minutes.
8. Centrifuged at 20,000 x g at 4°C for 30 minutes.
9. Once centrifuged, transferred clear supernatant to another centrifuge tube while avoiding all of the flakes on the sides and in the solution.
10. Centrifuged the tube again at 20,000 x g at 4°C for 15 minutes
11. While this ran, equilibrated the QIAGEN-tip by adding 4 mL of QBT to the QIAGEN-tip.
12. Added the clear solution (from step 10) to the QIAGEN-tip and allowed it to enter the resin by gravity flow
13. Next, added 10 mL of Buffer QC to the QIAGEN-tip and allowed to gravity drip.
14. Once that passed through, added 10 mL more of Buffer QC and allowed to flow through.
15. Then, added 5 mL of Buffer QF and allowed to flow through.
16. Added 3.5 mL of room temperature isopropanol to elute the DNA and mixed. Then centrifuged at 15,000 x g for 30 minutes at 4°C.
17. Carefully removed the supernatant making sure not to disrupt the clear pellet.
18. Added 2 mL of room-temperature 70% ethanol and centrifuged for 10 minutes at 15,000 x g at 4°C. Discarded the supernatant leaving as little liquid behind as possible, careful not to disrupt the clear pellet.
19. Air-dried the pellet for 20 minutes in the vent hood and redissolved in 100 μL of Buffer EB.

Results:

1. Will measure DNA concentration tomorrow.

Goal:

1. Our goal is to use midiprep to isolate each plasmid from the cultures of E.coli grown overnight. One plasmid should contain the Creilov gene and another plasmid should contain an ACS gene.

Protocol:

Follow protocol for qiagen midi prep

Purpose: Extract plasmid DNA (larger quantity) (Total time: 5 hours with 3 people)

QIAGEN Plasmid Midi Kit Protocol

Did you do your glycerol stocks?
1. Separated 500 mL of bacterial overnight culture into 2 sets of 5 separated 50 mL falcon tubes and centrifuged at 5,000 rpm for 15 minutes at 4°C.
2. Decanted supernatant.
3. Added 10 mL of Buffer P1 to two tubes, pipet mixed, and transferred to another tube.Mixed and transferred contents to the next tube with pelleted cells. Repeated until all tubes were combined.
4. Added 10 mL of Buffer P2 to the tube containing 10 mL of Buffer P1 and the combined resuspended ?pelleted cells and vigorously inverted 6 times.
5. Incubated at room temperat?ure for 3 minutes.
6. Added 10 mL of Buffer P3 (Buffer P3 and N3 is the same) and vigorously inverted 10 times.
7. Incubated on ice for 15 minutes.
8. Centrifuged at 5,000 rpm at 4°C for 30 minutes.
9. Once centrifuged, transfer clear supernatant to 2 high speed centrifuge tubes while avoiding all of the flakes on the sides and in the solution.
10. Centrifuged the tube again at 14,000 rpm at 4°C for 15 minutes
11. While this ran, equilibrated the QIAGEN-tip by adding 4 mL of QBT to the QIAGEN-tip.
12. Added the clear solution (from step 10) to the QIAGEN-tip and allowed it to enter the resin by gravity flow
13. Next, add 10 mL of Buffer QC to the QIAGEN-tip and allow gravity to drip.
14. Once that passed through, added 10 mL more of Buffer QC and allowed it to flow through.
15. Add 5ml buffer QF into the column to elute the DNA into a clean high speed plastic tubes. (USE GRAVITY FLOW)2
16. Precipitate DNA by adding 3.5ml room-temperature Isopropanol to the eluted DNA and mix. Centrifuge at 14,000 rpm for 30min at 4C.
17. Carefully removed the supernatant making sure not to disrupt the clear pellet.
18. Added 2 mL of room-temperature 70% ethanol and centrifuged for 10 minutes at 14,000 rpm at 4°C. Discarded the supernatant leaving as little liquid behind as possible, careful -not to disrupt the clear pellet.
19. Air-dried the pellet for 20 minutes in the vent hood and redissolved in 100 μL of Buffer EB.

Results:

1. Continued from step 18
2. The results we got for DNA Creilov was a concentration of 2 microliters per nanogram. The concentration of the DNA for the acs gene was a concentration of 4 microliters per nanogram. The concentrations were too low, so we will have to repeat the midiprep to isolate each of the two plasmids (Creilov gene and ACS gene).

Goal:

1. PCR reaction of ACS MP Midiprep on 7/26/22

Protocol:

1. 7 microliters of D.I. Water
2. 10 microliters of Dream Taq Master Mix
3. 1 microliter of VF primer
4. 1 microliter of VR primer
5. 1 microliter of ACS
6. ACS was run through the thermocycler according to the PCR protocol
7. VR, VF, and Dream Taq Master Mix were returned to the PCR box in the stand alone freezer. The ACS tube was returned to the clear micropipette holder in the stand alone freezer.

Results:

1. Will run gel on samples

Goal:

1. To transform iaaM, ami1, LPAAT, GPAT 1, GPAT 2, DGAT 1, DGAT 2 into E. coli

Protocol:

1. Combined 40 μL of electrically competent DH5a cells and 1 μL of ligated DNA to an Eppendorf tube.
2. Transferred the contents of the Eppendorf tube to a cuvette and lightly tapped the cuvette on the table to evenly distribute the contents and to get rid of air bubbles.
3. Placed the cuvette into the Bio-Rad MicroPulser and delivered the electric shock.
4. Immediately after, added 900 μL SOC medium to the cuvette and micropipette mixed the solution.
5. Transferred the solution from the cuvette to a shaker tube and placed in the shaker at 37°C at 200 rpm for 1 hour.
6. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.
7. Incubated plates at 37°C for at least 24 hours.

Results:

1. Expect to see growth tomorrow

Goal:

1. To run colony PCR on colonies on plates from 09.02.22

Protocol:

1. Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
2. Added 19 μL of the concentration cocktail into a PCR tube.
3. Using a 10 μL micropipette, touched the tip onto the selected colony and swirled around in the PCR tube.
4. Placed PCR tube in the thermocycler at the following generic settings:
   1. 95° C for 3:00 minutes
   2. 95° C for 1:00 minute
   3. 52° C for 1:00 minute *Annealing temperature varies depending on primer
   4. 72° C for 1:00 minute
   5. 30X (Go to Step 2)
   6. 72° C for 5:00 minutes
   Lid Temperature: 105° C

Results:

1. Picture of gel electrophoresis results

Goal:

1. to complete a mini prep to isolate each of the 7 Golden gate plasmids

Protocol:

Results:

• Recorded concentrations (recorded by Chloe)
• LB + amp iaaM: 0.65 ng/uL
• LB + AMP ami1: 0.60 ng/uL
• GPAT1: 12.5 μg/microliter
• DGAT 2: 0.3
• GPAT 1: 1.40
• GPAT: 2.065
• GPAT T1: 0.3
• 

  GPAT1: 12.5 μg/microliter
  

  ami1: 17.5 μg/microliter
• 

  DGAT1: 34.5 μg/microliter
  

  iaaM: 35.5 μg/microliter
• 

  DGAT2: 27.0 μg/microliter
  

  GPAT2: 23.0 μg/microliter
• 
  LPAAT: 14.0 μg/microliter

Goal:

1. Restriction digest on GPAT 1 & 2 and DGAT 1 & 2
2. Restriction digest on iaaM and ami1 to put into pSB1C3
3. PCR on midi prep on GPAT 1 & 2, DGAT 1 & 2 iaaM and ami1

Protocol:

Result:

Note

Goal:

Protocol:

1. Gel electrophoresis
2. Transformation
3. Restriction digest

Results:

Gel:

Transformation- successful, all parts grew on LB agar + chloramphenicol and did not grow on negative control

Goal:

1. Overnight culture
2. Gel electrophoresis

Protocol:

Results:

Overnight culture- done on 1A 1, 1I 1,15E 1, 13M 2
To be done on 1A 2, 15E 2, 16O 1, 16O 2, 13M 1
Gel electrophoresis-
Wells: 1kb ladder, GPAT 1, GPAT 2, empty, 1kb ladder, DGAT 1, DGAT 2
Camera function was not working, so I took the picture with my phone and the UV light blocking glasses.
GPAT 1, DGAT 1, DGAT 2 are clearly present. Gel extraction was attempted. GPAT 2 is not present.

Goal:

1. Midi prep
2. Overnight culture

Protocol:

Results:

Goal:

Protocol:

Results:

Goal:

Goal:

Protocol

1. Midi prep
2. Gel electrophoresis
3. Restriction digest
4. Overnight culture

Result

Goal:

Previous lab

Procedure

Results

Goal:

Protocol

Results

Goal:

Protocol

Results

Dr. Knoshaug Meeting Notes

• liquid tube of culture when it arrives
• Comes with literature for growth medium, provide light (PBR of some sort), growing in shake flasks in lighted incubator
• Shaking is good, algae will settle out
• PBR- provide CO2,
• Light incubators
• Really needs shaking
• Will settle, then cells on the bottom don’t get light and don’t grow
• Round bottom test tubes could work as well
• Cheaply mix using an aquarium pump with tube all the way to the bottom of the test tube, bubble air or CO2, would increase CO2 from air and provide mixing to keep algae floating
• Could work in flask, but big test tube would be best
• Set up in lighted incubator and bubble air
• Making algae growth media tends to precipitate, does not dissolve right
• Measure OD to determine growth, measure everyday or couple times a day since they grow quickly
• Growth levels off when self-shading starts to happen
• They try to keep them dilute to prevent self-shading and reduced growth rate
• Driving forces of making lipids is amount of light they can capture
• Inoculate culture at OD 0.1-0.2
• Monitor and see how fast OD gets up to 1
• High self-shading at 1
• Might have to try to keep co-cullture quite dilute
• Come in and take out half of culture and add media to keep culture diluted
• Avoid self-shading
• First determine how fast they are going to grow without co-cutulture and find upper density that they can reach
• Understand growth boundary and how fast they grow
• Add bacteria at OD of 0.3-0.4
• Could also add right when you add to your tubes
• Control tubes at 0.2 add water
• Experimental tubes at 0.2 add equivalent amount of E. coli
• See if growth takes off because of plant hormone, see if our experiment works
• Could be exposing algae to antibiotic
• Sometimes its a problem
• Can handle ampicillin
• CHLORAMPHENOCOL?
• Grow algae in antibiotic to maintain plasmid in E coli
• Grow algae in antibiotic from the start
• Unsure if chloramphenicol will have an affect on algae
• How many tubes fit in the incubator
• At least one tube and add antibiotic to see if algae can handle it
• Assuming it does, controls for co-culture, all will have antibiotic, some tubes without
• Negative has antibiotic and no e coli
• Experimental has antibiotic and e coli
• Also control group with e. coli modified with empty plasmid
• Start with 0.1 OD of algae and 0.1 of E. Coli, sum OD of 0.2, need room for things to grow
• Pellet and concentrate E. Coli to get just e coli, resuspend in water, take another OD, calculate how much to add to volume to get to 0.1 OD
• Every day, take OD, if it is too dense and use pipette to pull out half of volume, might even half to remove 75% to keep OD down, want it to get to 1 and then take it back down to get a good growth curve
• 100-250 mL tubes are good size for lipid extraction
• Need a fume hood for lipid extraction
• Using lipid philic dyes and look at with a florescent microscope
• Can be really hard to transform algae
• Hardest is that it takes a long time for algae to show up on plates, two weeks or longer
• Put efforts into making other parts of project to shine
• Make plates with genetcin and find appropriate concentration
• Lowest concentration that kills all other cells
• Can have contamination with untransformed algae
• Algae are good at getting around antibiotics
• Kanamycin doesn’t work in algae
• Linearizing plasmid could be more effective
• Algae recombine it into genome
• Can recombine with homologous arms and enter randomly (non homologous end-joining) or have 500-1000 with homology on either side of construct
• First thing to do is find antibiotic level that kills them
• Takes time to grow up, do comparisons, lipid extraction, etc
• Focus on co-culture first
• Set up growth curve and do thorough, detailed job to determine if E. Coli increases growth rate
• Lowest risk and biggest payoff for time
• Engineering of algae is 6 months-2 years
• Do it at least in triplicate
• Check ODs morning and night, better to do it often for dense data, not hourly but every 2-3 hours
• They are working with plant hormones for algae in outdoor trials
• Co-culture could be of very high value

Goal:

Protocol

Results

Goal:

Results

Goal:

Protocol:

Results