iGEM 2022 — Team ga-state-sw-jiaotong

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

[Date: 07/20/2022](#collapse-1)

### 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:

![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0720-1.png)
![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0720-2.png)

[Date: 07.22.22 | Name: Deva, Elizabeth, Yonatan](#collapse-2)

### 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?

[Date: 07/25/22 | Name: Deva, Kutura, Walter, and Zaria](#collapse-3)

### 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.

[Date: 7/26/22 | Names: Chlore, Deva, Christel](#collapse-4)

### 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).

[Date: 07/27/22 | Names: Kutura, Walter](#collapse-5)

### 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

[Date: 09/02/22 | Names: Elizabeth, Deva, Kutura, Aliyah](#collapse-6)

### 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

[Date: 09/03/22 | Names: Elizabeth, Deva, Kutura, Chloe, John, Manuel](#collapse-7)

### 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

[Date: 09/06/22 | Names: Chloe Benjamin, Kutura, Zaria, Yifei, Xucheng, Haobo](#collapse-8)

### Goal:

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

### Protocol:

QIAGEN - QIAprep Miniprep Handbook 4/98 -- QIAprep Spin Miniprep Kit
Protocol (harvard.edu)

### 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
* ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-1.png)

  GPAT1: 12.5 μg/microliter

  ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-2.png)

  ami1: 17.5 μg/microliter
* ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-3.png)

  DGAT1: 34.5 μg/microliter

  ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-4.png)

  iaaM: 35.5 μg/microliter
* ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-5.png)

  DGAT2: 27.0 μg/microliter

  ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-6.png)

  GPAT2: 23.0 μg/microliter
* ![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0906-7.png)

  LPAAT: 14.0 μg/microliter

[Date: 09/07/22 | Names: Aliyah, Sam, Jiatong](#collapse-9)

### 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:

##### PCR

* 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.
* Added 19 μL of the concentration cocktail into a PCR tube.
* Using a 10 μL micropipette, touched the tip onto the selected colony and swirled
  around in the PCR tube.
* 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

##### 30 μL Fast Digest Restriction Digest

* Prepared a Fast Digest concentration cocktail with the following proportions: 1
  μL Restriction Enzyme #1, 1 μL Restriction Enzyme #2, 3 μL of 10X Fast Digest
  Buffer, and 15 μL of diH2O.
* Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 10 μL
  of DNA
* Incubated at 37° C for 30 minutes

### Result:

Will run gel to view results tomorrow

### Note

PCR samples are still in thermocyler

[Date: 09/17/22 | Names: Elizabeth, Deva](#collapse-10)

### Goal:

Gel electrophoresis on PCR and miniprep samples, Transform and plate the promoter,
RBS, and terminator parts, restriction digest on GPAT and DGAT 1 & 2

Parts transformed from iGEM 2022 Kit Plate:

| Kit Location | Part | iGEM Repository |
| --- | --- | --- |
| 1A | promoter J23151 with GFP (BBa\_I20270) | [Link](http://parts.igem.org/Part%3ABBa_I20270) |
| 1I | composite part expressing GFP (BBa\_J364007) | [Link](http://parts.igem.org/Part%3ABBa_J364007) |
| 15E | RBS BCD12 (BBa\_K2680529) | [Link](http://parts.igem.org/Part%3ABBa_K2680529) |
| 13M | promoter J23119 | [Link](http://parts.igem.org/Part%3ABBa_J23119) |
| 16O | RBS (BBa\_Z0262) | [Link](http://parts.igem.org/Part%3ABBa_Z0262) |

##### Gel 1

Ami1-2
DGAT1
DGAT2
DGAT2
GPAT1
iaaM
iaaM3
GPAT1
GPAT2
GPAT1
GPAT2

### Protocol:

1. [Gel electrophoresis](https://docs.google.com/document/d/1z8TyyejmchBZUcV2w9I8ffA8WIw3u5TWNOZplcQsozU/edit)
2. [Transformation](https://docs.google.com/document/d/1PSs7QcTtasK6e3QDEvV_tpO3QB89fWzzvb3qo7UiBN4/edit)
3. [Restriction digest](https://docs.google.com/document/d/1AKjCohe34odYMXIp0QpYkkA_YeTqssPKjocn_8rCKM8/edit)

### Results:

Gel:

![](#)

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

![](#)

[Date: 09/18/22 | Names: Elizabeth, Zaria](#collapse-11)

### Goal:

Gel on restriction digest, overnight culture on cells transformed 09.17 (11)

1. [Overnight culture](https://docs.google.com/document/d/1gDMDRtr5s0-30ewBg9Dpi8j7zG4iZEXTyOyBZXPdXoo/edit)
2. [Gel electrophoresis](https://docs.google.com/document/d/1z8TyyejmchBZUcV2w9I8ffA8WIw3u5TWNOZplcQsozU/edit)

### Protocol:

[Ligation
Protocol with T4 DNA Ligase (M0202) (protocols.io)](https://www.protocols.io/view/ligation-protocol-with-t4-dna-ligase-m0202-95qpvorzv4o1/v3)

### 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-
![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/0918-1.png)
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.

[Date: 09/19/22 | Names: Jiatong, Yuqi, Deva](#collapse-12)

### Goal:

Midi prep on 1A 1, 1i 1, 1i 2, 15E 1, 13M 2; overnight culture on 1A 2, 15E 2, 16O 1,
16O 2, 13M 1

1. [Midi prep](https://docs.google.com/document/d/1hGEV7HL7LcH8QJamad92M9s187_yMnysy6FibkG2ovE/edit)
2. [Overnight culture](https://docs.google.com/document/d/1gDMDRtr5s0-30ewBg9Dpi8j7zG4iZEXTyOyBZXPdXoo/edit)

### Protocol:

[Ligation
Protocol with T4 DNA Ligase (M0202) (protocols.io)](https://www.protocols.io/view/ligation-protocol-with-t4-dna-ligase-m0202-95qpvorzv4o1/v3)

### Results:

| Part | Concentration | A260/280 |
| --- | --- | --- |
| 1i 2 A | 21 | 1.913 |
| 1i 2 B | 25.5 | 1.839 |
| 1i 2 C | 290 | 1.758 |
| 1i 2 D | 51.5 | 1.839 |
| 1i 2 E | 210 | 1.808 |
| 1A 1 A | 115 | 1.817 |
| 1A 1 B | 95 | 1.833 |
| 1A 1 C | 12 | 2.000 |
| 1A 1 D | 22 |  |
| 15E 1, 13M 2 | Too low to be measured |  |

[Date: 09/21/22 | Names: Jiatong, Yuqi, Deva](#collapse-13)

### Goal:

Restriction digest on kit plate parts and IAA parts, ligate RBS and IAA parts together

### Protocol:

[Restriction digest](https://docs.google.com/document/d/1AKjCohe34odYMXIp0QpYkkA_YeTqssPKjocn_8rCKM8/edit)

### Results:

| 2022 Kit plate 1 | Part | Enzymes |
| --- | --- | --- |
| 1A | promoter J23151 with GFP (BBa\_I20270) | SpeI, PstI |
| 1i | composite part expressing GFP (BBa\_J364007) | EcoRI, PstI |

[Ligation](https://docs.google.com/document/d/1OVKntsC46Nf9fInehZuFUP6h2sMxXF1A_MXhRNlBH5c/edit?usp=sharing)
on 15E and iaaM, 15E and ami1, 16O and iaaM, 16O and ami1

[Date: 09/22/22 | Names: Jiatong](#collapse-14)

### Goal:

[Ligation](https://docs.google.com/document/d/1OVKntsC46Nf9fInehZuFUP6h2sMxXF1A_MXhRNlBH5c/edit?usp=sharing)
on 15E iaaM and ami1, 16O iaaM and ami1 (ligating together parts with the same RBS)
Will run gel to confirm success

[Date: 09.23.22 | Names: Aliyah, Deva, John, Chloe, Rainbow, Elizabeth](#collapse-15)

### Goal:

Midi prep on 1I 1, 1I 1,, 1I2 1A 1; gel electrophoresis on mini prep from last week and
restriction digest 09.22, overnight culture on iaaM and ami1

### Protocol

1. [Midi
   prep](https://docs.google.com/document/d/1hGEV7HL7LcH8QJamad92M9s187_yMnysy6FibkG2ovE/edit?usp=sharing)
2. [Gel
   electrophoresis](https://docs.google.com/document/d/1z8TyyejmchBZUcV2w9I8ffA8WIw3u5TWNOZplcQsozU/edit?usp=sharing)
3. [Restriction
   digest](https://docs.google.com/document/d/1AKjCohe34odYMXIp0QpYkkA_YeTqssPKjocn_8rCKM8/edit?usp=sharing)
4. [Overnight
   culture](https://docs.google.com/document/d/1gDMDRtr5s0-30ewBg9Dpi8j7zG4iZEXTyOyBZXPdXoo/edit?usp=sharing)

### Result

| Part | Concentration | A260/280 |
| --- | --- | --- |
| 1A 1 | 735 | 1.878 |
| 1i 1 | 17 | 1.842 |
| 1i 2 | 760 | 1.831 |
 Still to be done:   Midi prep on 15E, 13M, 16O   Restriction digest on | | || 15E | RBS BCD12 (BBa\_K2680529) | SpeI, PstI |
| 13M | promoter J23119 | SpeI, PstI |
| 16O | RBS (BBa\_Z0262) | SpeI, PstI |
| iaaM |  | XbaI, PstI |
| ami1 |  | XbaI, PstI |

[Date: 09/24/22 | Names: Deva, John, Chloe](#collapse-16)

### Goal:

[Ligation](https://docs.google.com/document/d/1OVKntsC46Nf9fInehZuFUP6h2sMxXF1A_MXhRNlBH5c/edit?usp=sharing)
on 15E iaaM and ami1, 16O iaaM and ami1 (ligating together parts with the same RBS)
Will run gel to confirm success

### Previous lab

We left iaaM and ami1 overnight cultures in the shaker to do midi preps today, but there was no pellet in the overnight cultures, so we had to go ahead and make newly transformed plates

### Procedure

[Transformation](https://docs.google.com/document/d/1PSs7QcTtasK6e3QDEvV_tpO3QB89fWzzvb3qo7UiBN4/edit?usp=sharing)

### Results

Left iaaM and ami1 plates in the 4 degrees fridge. We need to do overnight cultures on them on saturday
(Parts registry can be found here)
Midi prep concentrations of DNA from Thursday and Friday:

| Part | Concentration | A260/280 |
| --- | --- | --- |
| 1i 2 A | 21 | 1.913 |
| 1i 2 B | 25.5 | 1.839 |
| 1i 2 C | 290 | 1.758 |
| 1i 2 D | 51.5 | 1.839 |
| 1i 2 E | 210 | 1.808 |
| 1A 1 A | 115 | 1.817 |
| 1A 1 B | 95 | 1.833 |
| 1A 1 C | 12 | 2.000 |
| 1A 1 D | 22 | Too low to be measured |
| 15E 1, 13M 2 | Too low to be measured | Too low to be measured |

[Date: 09/26/22-09/27/22 | Names: Deva, Elizabeth](#collapse-17)

### Goal:

Record concentrations of midi prep on iaaM and ami1
Overnight culture on pBI121

### Protocol

Midi prep
Overnight culture

### Results

| Part | Concentration (μg/ml) | A260/280 |
| --- | --- | --- |
| Ami1 - a | 97.5 | 1.757 |
| Ami1 - b | 45 | - |
| Ami1 - c | 113 | 1.957 |
| Ami 1 - d | 128 | 2.040 |
| Ami1- e | 95 | 1.810 |
| Ami1-f | 102 | 1.152 |
| Iaam 3 | 97.5 | 1.773 |
| Iaam 4 | 115 | 1.533 |
| Iaam 5 | 80.0 | 2.286 |

[Date: 09/28/22 | Names: Deva](#collapse-18)

### Goal:

Midi prep on PBI121

[Restriction digest on iaaM and ami1](https://docs.google.com/document/d/1AKjCohe34odYMXIp0QpYkkA_YeTqssPKjocn_8rCKM8/edit?usp=sharing)

[Ligate with promoter](https://docs.google.com/document/d/1OVKntsC46Nf9fInehZuFUP6h2sMxXF1A_MXhRNlBH5c/edit?usp=sharing)

### Protocol

Midi prep
Restriction digest
Ligation

### Results

| Part | Concentration (μg/ml) | A260/280 |
| --- | --- | --- |
| pBI121 1 | 32.5 | 0.650 |
| pBI121 2 | 195 | 1.418 |
| pBI121 3 | 670 | 1.624 |

### 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

[Date: 10/01/22 | Names: Kutura, John, Elizabeth, Deva, Chloe](#collapse-19)

### Goal:

Midi prep on iaaM and ami1 with 1I2 promoter, colony PCR on iaaM and ami1

### Protocol

Absorbance measured in Standard mode with a concentration of 50.0 and a pathlength of 10 mm and factor of 120

### Results

| Sample | OD at 260nm | Concentration at 260 nm | OD at 280 nm | Concentration at 280 | Purity Ratio 260/280 |
| --- | --- | --- | --- | --- | --- |
| 1 ami 1 (1I2) | 0.891 | 107 | 0.098 | 11.8 | 9.09 |
| 2 ami 1 (1I2) | 2.240 | 269 | -0.106 | None | -21.1 |
| 3 ami 1 (1I2) | -0.049 | None | -0.100 | None | 0.490 |
| 4 ami 1 (1I2) | -0.037 | None | -0.108 | None | .343 |
| 5 ami 1 (1I2) | 0.028 | 3.360 | -0.157 | None | -0.178 |
| 1 iaam (1I2) | 0.005 | .600 | -0.022 | None | -.227 |
| 2 iaam (1I2) | 0.057 | 6.840 | 0.088 | 10.6 | .648 |
| 3 iaam (1I2) | 0.096 | 11.5 | 0.004 | 0.480 | 24.0 |
| 4 iaam (1I2) | -0.001 | none | 0.022 | 2.640 | -.045 |
| 5 iaam (1I2) | -0.040 | none | 0.052 | 6.240 | -.769 |

| Number on PCR Tube | Part |
| --- | --- |
| 2 | iaaM 1I2 1 |
| 3 | A ami1 1I2 1 |
| 4 | A ami1 1I2 2 |
| 5 | B ami1 1I2 1 |
|  |  |

| Part | Restriction Enzyme |
| --- | --- |
| LPAAT for pBI121 | EcoRI, SpeI |
| LPAAT for DinoIII | XbaI, SpeI |
| For ligation |  |
| DGAT |  |

[Date: 10/06/22 | Names: Kutura, Chloe Benjamin, Deva, Elizabeth, Aliyah](#collapse-20)

### Goal:

Protein purification

### Results

| Sample | OD REading at 600 nm |
| --- | --- |
| ami | 0.774 |
| 1 ami1+iaam | 0.674 |
| 1 iaam | 2.145 |
| 2 iaam+ami1 | 637 |
| 2 iaam | 2.256 |

[Date: 10/11/22 | Names: Deva and Kutura](#collapse-21)

### Goal:

Colony PCR ami1 + promoter and iaam + promoter and OD

### Protocol:

Colony PCR

### Results

![](https://static.igem.wiki/teams/4493/wiki/wiki/notebook/1011-1.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

### Abstract

Greenhouse gas emissions arising from the combustion of
fossil fuels are major contributors to climate change. Biofuels offer a carbon-neutral
alternative: microalgae can be used to generate  biofuels by absorbing  carbon
dioxide for photosynthesis. Our goal is to create a synthetic biology system that will
increase lipid production from microalgae to serve as an intermediate in chemical reactions
needed to create biofuels. Specifically, we will create a co-culture between
*C. minutissima*microalgae and *E. coli* bacteria. To increase *C. minutissima*’s growth rate, *E. coli* will be genetically modified to produce indole-3-acetic
acid (IAA). *C. minutissima* will be genetically modified to express
glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltrans- ferase
(LPAAT), and diglyceride acyltransferase (DGAT) to increase lipid production. We hypothesize
that this modified co-culture will result in a dense algae population with high lipid
content, providing ideal parameters for biofuel production.

### Engineering

Through the project, Synknlorum, we aimed to produce a
co-culture product between the organisms *E.
Coli* and *C. Minutissima* that were synthetically modified to maximize lipid
production. The lipids produced by the algae could be used to produce biofuels on an
industrial scale.

### Design

For several decades fossil fuels have led the market as the
main supply of energy for lighting, heating, and transportation. [1] As a non-renewable
resource, the increasing population and need for more resources put fossil fuels at risk of
scarcity. Burning these fossil fuels also contributes to 85% of Carbon dioxide released into
the atmosphere. Carbon dioxide scatters infrared radiation, inducing the greenhouse effect
and accelerating global warming and climate change.  More sustainable and renewable
alternatives to fossil fuel production have to be considered. Algal biofuel technology
offers a more sustainable alternative. Lipid-producing microalgae, *Chlorella minutissima,* has shown promise in increased neutral lipid production and
biodiesel quality when
 co-cultured with the
bacteria E. Coli. Therefore, we wanted to recreate this co-culture in our lab after
synthetically modifying the C. Minutissima with genes for lipid-producing  enzymes,
LPAAT, DGAT, GPAT, and the E. Coli with a plant growth hormone, IAA. The two organisms would
be grown together in a co-culture. We hypothesized that the algae in the co-culture would
have an increased optimal density and lipid production compared to an algae without the
co-culture. The finalized
 product would be
sent to the NERL to produce biofuels using their more advanced technology.

### Build

We chose the microalgae, *Chlorella minutissima* due to its ability to grow in varied conditions, making it
ideal for growing in our lab. *E.
Coli* was the ideal bacterium to use in
the co-culture as it has an incredible amount of research supporting it and has been used in
similar co-cultures.

### E. Coli

The engineered E. Coli was modified using two genes in the
IAM pathway, tryptophan 2-monooxygenase (iaaM) and amidase 1 (ami1), to produce the plant
growth hormone Indole-3-acetic acid (IAA). The genes were all designed using the SnapGene
software and synthesized via the IDT sponsorship that iGEM provided us.

![](https://static.igem.wiki/teams/4493/wiki/wiki/engineering/1.jpg)![](https://static.igem.wiki/teams/4493/wiki/wiki/engineering/2.jpg)

### C. minutissima

The *C.
minutissima* was to be genetically
modified with enzymes involved in lipid production, which included a glycerol-3-phosphate
acyltransferase (GPAT), lysophosphatidic acid acyltrans- ferase (LPAAT), and diglyceride
acyltransferase (DGAT). These gene constructs were also designed in  SnapGene and
synthesized by  IDT. The constructs were ligated onto a DinoIII plasmid from our
previous iGEM project and induced using the CAMV promoter to allow for expression in
algae.

### Test

### Verification of constructs

A colony PCR was performed on the colonies that we grew
overnight, and the results of the gel can be read below. The genes were also sent for
sequencing to verify that they contained our genes of interest.

### Western Blotting

We performed protein extraction and isolation on our
transformed cultures. However, we chose not to use our samples for the western blotting as
our technique while handling the protein isolation was poor. In the future, we aim to
perfect our technique and proceed to perform a western blot on our two ligated
plasmids.

### Plasmid Transformations

We
picked a colony from each plate and the culture out overnight in our shaker to perform midi
preps the next day. We also did a colony PCR using colonies from the plate in order to
verify the presence of our ligated product. The readings on the midi prep and the gel from
the colony PCR were good so we were certain that we should proceed to perform a protein
extraction and western blot to detect any protein expression.

### Proposed Implementation

After we have successfully proven that co-culture between C.
minutissima and E. coli leads to increased amounts of lipids in the microalgae, we plan to
market the project idea to the [National Renewable Energy Laboratory
(NREL)](https://www.nrel.gov/). We have discovered through our
human practice interaction with them that they are interested in the co-culture methods to
increase lipid production, so
 our project
would hopefully be something they look forward to taking on.

### Sources

     1.Hannon M, Gimpel J, Tran M, Rasala B, Mayfield S. Biofuels
from algae: challenges and potential. Biofuels. 2010 Sep;1(5):763-784. doi:
10.4155/bfs.10.44. PMID: 21833344; PMCID: PMC3152439.

2.Higgins, B. T., & VanderGheynst, J. S. (2014). Effects
of Escherichia coli on mixotrophic growth
of
Chlorella minutissima and production of biofuel precursors. PloS one, 9(5), e96807.
<https://doi.org/10.1371/journal.pone.0096807>

3.Varela Villarreal J, Burgués C, Rösch C. Acceptability of
genetically engineered algae biofuels in Europe: opinions of experts and stakeholders.
Biotechnol Biofuels. 2020 May 22;13:92. doi:
10.1186/s13068-020-01730-y. PMID: 32489422; PMCID:
PMC7245023.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## **Experimental Plan**

###

### Week of July 18th

##### 07.20 Wednesday

Transform acs and CreiLOV
Restriction digest on acs and CreiLOV
Meet with Dr. Chin

##### 07.21 Thursday

Overnight cultures

##### 07.22 Friday

Meet with Toulouse iGEM Team
Midi-prep on overnight cultures
Make glycerol stocks from overnight cultures

### Week of July 25th

##### 07.25 Monday

Midi-prep of acs and CreiLOV

##### 07.26 Tuesday

Midi-prep on acs and CreiLOV

##### 07.27 Wednesday

PCR of acs

### Week of August 29th

##### 09.02 Friday

Transform E. coli with IDT parts (iaaM, ami1, LPAAT, DGAT 1, DGAT 2, GPAT 1, GPAT 2) Plate
Plasmid = pUCIDT-AMP Genes = iaaM, ami1, LPAAT, DGAT1, DGAT2, GPAT1, GPAT2

##### 09.03 Saturday

Run PCR reaction on colonies with IDT parts

### Week of September 5th

##### 09.05 Monday

Overnight cultures
Run gel with DNA from PCR from 09.03

##### 09.06 Tuesday

Midi preps on overnight cultures

##### 09.07 Wednesday

Restriction digest on GPAT 1 & 2 and DGAT 1 & 2
Restriction digest on iaaM and ami1 to put into pSB1C3
Restriction digest on promoter, RBS, and terminator from kit plates for expression in E coli

##### 09.08 Thursday

PCR on midi prep from 09.06
Ligate GPAT 1 & 2 and DGAT 1 & 2
Ligate genes for IAA operon (order: promoter, RBS, iaaM, RBS, ami1, terminator)

##### 09.09 Friday

Gel on midi prep PCR from 09.08
Debrief on experimental progress

##### 09.10 Saturday

- digest parts 2022 kit plate 1
[Promoter in PSB1C3SA plasmid](http://parts.igem.org/Part%3ABBa_J23111)
[Promoter + GFP in PSB1C3 (could not find only the promoter in PSB1C3)](http://parts.igem.org/Part%3ABBa_I20270)
[RBS in pSB1C3C](http://parts.igem.org/Part%3ABBa_K2680531)
[Terminator in pSB1C3SD](http://parts.igem.org/Part%3ABBa_J428069)
- Digest parts 2021 kit plate 1 (I think we should use these if we can find the kit plates)
[Terminator in PSB1C3](http://parts.igem.org/Part%3ABBa_K731722)
[Promoter in pSB1C3](http://parts.igem.org/Part%3ABBa_K733001)
[RBS in pSB1C3](http://parts.igem.org/Part%3ABBa_K545666)

### Week of September 12th

##### 09.12 Monday

Couldn’t find gel stain :(
Run gel with DNA from PCR from 09.03

##### 09.13 Tuesday

Transform promoter, RBS, and terminator parts into E. coli
Restriction digest on a plasmid containing GUS gene from Synbiodinium project
Ask Dr. Brewer about steps to Western Blot
Look for tryptophan, Western Blot equipment, and competent cell stock in the lab

##### 09.14 Wednesday

Overnight culture on promoter, RBS, terminator
Make LB Broth
The Gel stain has arrived in the lab :)

### Week of September 12th

##### 09.12 Monday

Couldn’t find gel stain :(
Run gel with DNA from PCR from 09.03

##### 09.13 Tuesday

Transform promoter, RBS, and terminator parts into E. coli
Restriction digest on a plasmid containing GUS gene from Synbiodinium project
Ask Dr. Brewer about steps to Western Blot
Look for tryptophan, Western Blot equipment, and competent cell stock in the lab

##### 09.14 Wednesday

Overnight culture on promoter, RBS, terminator
Make LB Broth
The Gel stain has arrived in the lab :)

##### 09.15 Thursday

MIDI (not mini, should have 500 ml LB) prep on the promoter, RBS, terminator
[Protocol](https://docs.google.com/document/d/1hGEV7HL7LcH8QJamad92M9s187_yMnysy6FibkG2ovE/edit?usp=sharing)

##### 09.16 Friday

Restriction digest on promoter, RBS, terminator
Ligate IAA sequence (order: promoter, RBS, iaaM, RBS, ami1, terminator)

##### 09.17 Saturday

Transform IAA sequence into E. coli (using rosetta cells for expression, not the usual electrocompetent cells)
Ask Eric what data is needed for the IAA model

##### 09.18 Sunday

Overnight culture on 1A 1, 1i 1,15E 1, 13M 2
Gel electrophoresis and gel extraction on DGAT 1 & 2 and GPAT 1 & 2

### Week of September 19th

##### Monday 09.19

Midi prep on 1A 1, 1i 1,15E 1, 13M 2
Overnight culture on 1A 2, 15E 2, 16O 1, 16O 2, 13M 1

##### Wednesday 09.21

Restriction digest on 1A and 1i

##### Thursday 09.22

Midi prep on \_\_\_\_\_\_\_\_\_\_\_.

##### Friday 09.23

Midi prep on 1A1 and 1I1
Restriction digest on plasmids from 09.22
Ligate promoter part into plasmids

### Week of September 19th

##### Monday 09.26

Midi prep on iaaM and ami1
Overnight culture on pBI121

##### Tuesday 09.27

Record concentrations of results on midi prep on iaaM and ami1
No midi prep needed for pBI121 (algae plasmid)
Restriction digest on iaaM and ami1
Ligate iaaM and ami1 with promoter
[Meeting with Dr. Knoshaug](https://docs.google.com/document/d/1dxSdwybcJZtmvsnggfd3qBeerbRWyp1CC5zAjXhJEqQ/edit?usp=sharing)

##### Wednesday 09.28

Transform complete iaaM and ami1 construct into E. coli
Check lab equipment for Western blot (transfer machine and shaker)

##### Thursday 09.29

Overnight culture on iaaM and ami1 constructs
Colony PCR on plates from 09.28

##### Friday 09.30

Start Western blot (no western blot done today)

##### Saturday 10.01

Finish Western blot

![](https://static.igem.wiki/teams/4493/wiki/wiki/experiment.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

### **Parts Construction**

For this project, a total of 13 parts were created, 11 basic parts and
2 composite parts. IDT and Twist Bioscience synthesized the basic parts, and the composite
parts were assembled by restriction digestion and ligation in our lab. Each part is
compatible with the BioBrick RFC[10] standard. Genes for the TAG lipid production pathway
and the IAA pathway were codon optimized for C. minutissima and E. coli, respectively, and a
6xHis tag was added to the end of each coding sequence for convenient protein
isolation.

However, due to the codon optimization process, many genes had high
complexity and could not be chemically synthesized. To solve this problem, sequences
were manually modified to maintain the same codons
and as much codon optimization as possible while reducing repeat
sequences.

Some genes, such as DGAT and GPAT, were too complex to synthesize.
These genes were synthesized in two parts based on the naturally existing PvuII site near
the middle of the gene. This ensured the parts could be synthesized and that our lab could
ligate the parts together without having to purchase new materials.

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/1-result.png)![](https://static.igem.wiki/teams/4493/wiki/wiki/result/2-result.png)

### *Escherichia coli*

The
genes that we wanted to tranform into E. coli were the iaam and ami1 genes which code for
the protein, tryptophan 2-monooxygenase (iaaM) and amidase 1 (ami1) respectively. We used
promoters, RBS, and terminators found in the iGem 2022 DNA kit plate. The two that we
got the highest concentration of DNA from our midi
preps were pSB1C3 plasmids with promoter J23100 (BBa\_J364007) and J23151 (BBa\_I20270). These
were later used to ligate our genes of interest and transform into electrocompetent E. coli
cells.

pSB1C3 +
iaam + J23151 Plasmid Construction

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/3-result.jpg)

pSB1C3
+ami1 + J23100 Plasmid

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/4-result.jpg)

Construction

The
parts were ligated as shown above, and transformed into electrocompetent Dh5-a
*E. coli* cells.
They were then plated on Luria Broth agar plates with chloramphenicol added to it.
The colonies were grown in the incubator
overnight.

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/5-result.jpg)![](https://static.igem.wiki/teams/4493/wiki/wiki/result/6-result.jpg)

We
picked a colony from each plate and the culture out overnight in our shaker to perform midi
preps the next day. We also did a colony PCR using colonies from the plate in order to
verify the presence of our ligated product. The readings on the midi prep and the gel from
the colony PCR were good so we were certain that we should proceed to perform a protein
extraction and western blot to detect any protein expression.

ami1 +
J23151 in pSB1C3 midi prep results:

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/7-result.jpg)![](https://static.igem.wiki/teams/4493/wiki/wiki/result/8-result.jpg)

iaam +
J23100 in pSB1C3 midi prep results:

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/9-result.jpg)![](https://static.igem.wiki/teams/4493/wiki/wiki/result/10-result.jpg)

Colony
PCR on iaam + J23100 and ami1 + J23151 in pSB1C3

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/11-result.jpg)

The
bands were in the base pair range that we were expecting to see so we went ahead with our
protein extraction. However, the procedure was relatively new to us, so human error
prevented us from attaining strong promising results during our protein
purification.

### *Chlorella minutissima*

We chose
two plasmids that we had used in our earlier iGem projects, PBI121 and DinoIII plasmids and
performed a gel extraction to gain. The homologous arms present in our DinoIII plasmid match
our algae, *C. minutissima*, this makes it ideal for expression in our algae. The ligated LPAAT,
DGAT, and GPAT parts will be inserted into both plasmids and transformed into the
algae.

![](https://static.igem.wiki/teams/4493/wiki/wiki/result/12-result.jpg)![](https://static.igem.wiki/teams/4493/wiki/wiki/result/13-result.jpg)

{% endblock %}