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Notebook

Project day by day.

Nitrogen

Left side of timeline

Phosphorus

Right side of timeline

April 5 - July 6, 2023

Brainstorming, project selection, ordering supplies, outreach

July 6, 2023

Goal: Determine a project, order all necessary supplies, communicate with stakeholders and environmental experts for advice
We organized our team, researched local problems, decided on Utah Lake as our target, and researched main problems and potential solutions. We researched and found that nutrient loading in Utah Lake was a main problem, and decided to decrease phosphorus and nitrogen in the lake. We chose C. reinhardtii as our chassis, picked genes we wanted to express/suppress, ordered media, genetic parts, and the Plant and Chlamy Moclo kits. We also communicated with various local experts and stakeholders with regards to Utah Lake, environmental preservation, water treatment, and toxic algal blooms.

July 6 - August 23, 2023

PSR1: (Starvation response regulator) preparation and ordering
Chlamydomonas reinhardtii already has the natural ability to accumulate and store excess phosphate (PO43-) into Polyphosphate as a Phosphate reserve to sustain growth and critical metabolic functions under low exogenous Phosphate or during conditions of phosphate depletion.
PSR1 is a global transcriptional regulator of phosphorus deficiency responses, over expression of this gene induces storage metabolite biosynthesis, increasing the abundance of the P homeostatic components and, consequently, increases P uptake into the cell, even under P-replete conditions. This indicates that the P starvation response can be deregulated, allowing excessive P accumulation even when not needed by the cell.

July 6, 2023

Ligation of the Level 0 plasmids for AMO, HAO, and nosZ

July 6, 2023

Goal: To assemble Level 0 plasmids for AMO, HAO, and nosZ
CDS sequences of AMO, HAO, and nosZ were codon-optimized and domesticated by removing cut sites for SapI, BsaI, and BbsI and synthesized by GenScript.
We assembled 20 uL reactions using the Golden Gate Assembly Protocol with SapI, using BBa_J04450 as the recipient plasmid.

July 7, 2023

Transformation of E. coli

July 7, 2023

We transformed 2 uL of each ligation reaction into NEB 10Beta cells using the NEB Protocol, and plated 50 uL of bacterial suspension onto selective plates.

July 8, 2023

Inoculation of liquid cultures

July 8, 2023

Three to four colonies from each plate were inoculated into liquid cultures and grown at 37C overnight.

July 12, 2023

Miniprep of L0 plasmid

July 12, 2023

We miniprepped the L0 AMO, HAO, nosZ plasmids. A red colony was also miniprepped as a negative control.

Gene ng/mL 260/280 260/230 
HAO 1 52 1.78 1.75 
HAO 2 52.3 2.03 2.39 
HAO 3 18.7 2.81 -5.35 
HAO 4 59.6 2.02 2.54 
NOSZ 1 43.9 1.95 1.37 
NOSZ 2 35.2 1.69 4.04 
NOSZ 3 21.9 1.26 5.58 
NOSZ RED 1a1.8 6.95 -71.95 
AMO 1 31.2 2.18 3.72 
AMO 2 168.3 1.98 0.69 
AMO 3 44.2 2.05 2.34 

July 13, 2023

Diagnostic digest

July 13, 2023

Lane 1 is AMO clone 3. Lane 2 is Ladder. Lanes 3-5 are HAO clones 1-3. Lane 6 is empty vector control. Lanes 7-9 are nosZ clones 1-3. Lanes 10-11 crnpt level 1 plasmids cut with Bam/Nde. Lane 12 is empty. Lane 13-15 are AMO clone 1, 1 again, and 2.

July 18, 2023

Sequencing of L0 plasmids

July 18, 2023

Submitted 500 ng of each plasmid with sequencing primer to Psomagen.
Golden Gate L1 Plasmid Assembly for HAO and AMO
Procedure: Jacob and Makensie used the ligation template and golden gate assembly to create L1 plasmids for HAO (flag) and AMO (His). Clone #1 from L0-HAO and L0-AMO was used for the assembly. Reference the ligation template for amounts used and how each L1 plasmid was assembled.

July 19, 2023

Transformation of L1-HAO and AMO ligation reactions

July 19, 2023

2 uL of each L1 ligation transformed into NEB10beta cells using standard protocol

July 19, 2023

Diagnostic digest of L0-nosZ plasmid

July 19, 2023

We performed a diagnostic digest of the L0 nosZ minipreps 1-3 and red (negative control) because because the primary digest that we did on 7.10.23 was uninterpretable. A better digest for L0-nosZ would be a NotI digest. The vector would yield 2046bp + 1122bp and the nosZ clone would be 2046bp + 1662b.

  • NOS Z 1: Results were not promising
  • NOS Z 2: As expected for successful insertion of nosZ.
  • NOS Z 3: As expected for successful insertion of nosZ.
  • Red control: As expected for empty vector.

The digest supports the successful construction of the L0-nosZ plasmid for colonies #2 and 3. Colony 2 was later used for construction of the Level 1 plasmid.

July 20, 2023

Re-streaking of L0 transformants

July 20, 2023

Since we didn't have enough culture for further sequencing we streaked our HAO, AMO, & NOSZ 2 & 3 tubes onto plates. We didn't streak NOS Z 1 since its gel result didn't look very promising.
We also did a re-streak of the L1 HAO (47742 Flag Tag) and AMO (47751 His Tag) plasmids because there was a very low concentration of white colonies.

July 25, 2023

Inoculation of L0-HAO liquid cultures to create more plasmids

July 25, 2023

We decided to focus on sequencing of L0-HAO plasmid for the next time period. We inoculated 3 tubes of L0-HAO transformants and incubated at 37C overnight with shaking at 250 rpm.

July 26, 2023

Miniprep of Level 0 HAO clones

July 26, 2023

Due to low yields, two different groups of students miniprepped the liquid cultures to see if user error was causing the problem.
Mini prep results
Readings L0 HAO 1-3
Group A:

Sample ng/uL A260/270 260/230 DNA H2O
HAO 1 56.0 1.8 0.97 - -
HAO 2 134.0 1.65 0.83 4uL 5uL
HAO 3 90.8 1.81 1.08 6uL 3uL


Group B
ng/uL A260/270 260/230 DNA H2O
HAO 1 132.0 1.83 1.41 4uL 5uL
HAO 2 53.6 1.77 1.17 - -
HAO 3 82.6 1.85 1.23 - -

Looking at the Level 1 AMO and HAO colonies we re-streaked on 7/20/23, we saw that they looked similar to the original streaks. Both original & re-streaked plates had too many blue, unsuccessful colonies, so we took some colonies from both re-streaked plates and streaked them onto new plates.
We went back to the original L1-HAO and AMO ligation reactions and redid the transformation.

July 31, 2023

Picking more colonies

July 31, 2023

L0 NOS 2,3, and L0 AMO 1,2,3, colonies were picked to prepare cultures for sequencing on 8.1.23

August 1, 2023

NOS & AMO Preps, Gel Electrophoresis, and Sequencing Prep

August 1, 2023

Performed a HindIII digest for the L1 plasmids.

Initially we thought the digest results were as expected, but later (end of September), closer inspection lead us to conclude that the HAO digests had an extra band; the AMO plasmid digest patterns were uninterpretable.
We prepared L1 HAO to send in for sequencing. This sequencing reaction failed, which we later determined was due to the fact that the plasmids were incorrect and contained unknown vector sequence.
We picked 4 new L1-AMO colonies. - AMO 4,5,6,7

August 3, 2023

More L1-AMO Minipreps

August 3, 2023

We did minipreps for L1-AMO 4,5,6,7,8,9,10,11, and the blue control which were picked on 8.1.23. to get better AMO with better spec results.

August 8, 2023

L1-AMO Diagnostic Digests with NdeI

August 8, 2023

L1 digests of AMO 4-11
AMO 4-11 and blue results:

Key: ladder, blue, 11,10,9,8,7,6,5,4,3,2,1
The columns were ordered with blue at the left after the ladder, then AMO 11 down to 4 at the right.
They have multiple bands for some reason.
AMO 6 and 5 don't have DNA since the digests weren't done properly.
Due to the repeated failure of our diagnostic digests, we decided to send out L1 AMO 7 and 8 for sequencing.
Subsequent analysis of the sequencing results supported the successful build of the L1-AMO-His plasmid.

August 22, 2023

L1 cloning of nosZ-clover

August 22, 2023

Elise set up an L1 ligation reaction for nosZ.

August 23, 2023

Transformation of L1-nosZ-clover ligation reaction

August 23, 2023

Blaise transformed 3 uL of the ligation reaction into 50 uL of NEB 10-beta cells and plated on LB+amp+IPTG+Xgal plates.

August 24, 2023

Cloning of Psr1

August 24, 2023

We got the L0-Psr1HA(no stop) plasmid, which we recieved from another lab (main thing we wanted to obtain was the Psr gene). Then we set up a ligation to create an L1 plasmid:

L1 plasmid: PPSAD (promoter + 5' UTR) (slot A1-B2), PsrHA(slot B3-B4), Clover (i2)-Strep (C-ter, CDS) (slot B5), TPSAD (3' UTR + Ter) (slot B6-C1) put together using Chamy MoClo toolkit.

August 24, 2023

Colony pick

August 24, 2023

Zenos, Blaise and Caroline picked 4 white and 1 blue colony and inoculated each into 3 mL LB + amp, placed on 37C shaking incubator O/N at 250 rpm. Also streaked each colony onto a new plate.

August 25, 2023

High Efficiency Transformation of L1 plasmid in E. coli.

August 24, 2023

High Efficiency Transformation was performed with the L1 PSR clover Ligation, using NEB 10-beta competent E. coli cells. Then 100 uL and 50 uL of cells were added to ampicillin plates.
Dr. D. removed plates on 8/26; a good number of white colonies suggesting successful ligation reactions.
On 8/27/23, Dr. D picked 4 white (have gene) and 1 blue colony (control) and inoculated each into 3 mL LB + 100 ug/mL amp and placed in 37-degree incubator shaking at 250 rpm.

August 25, 2023

L1 nosZ-clover minipreps and diagnostic digest

August 25, 2023

Audrey and Elise miniprepped the nosZ samples. 5 is a negative control. 500 ng of each plasmid was subsequently digested with SacI. Expected fragment sizes are 8 kb and 1.5 kb.

Sample ng/uL A260/270 260/230
1 218.4 1.88 2.27
2 265.5 1.88 2.18
3 229.7 1.88 2.25
4 173.5 1.80 2.32
5 144.2 1.86 2.10


The data support the successful construction of the L1-nosZ-clover plasmid.

August 28, 2023

L1 Plasmid mini preps

August 28, 2023

The plasmid mini preps were performed from the previous picked colonies:
-L1 PsR HA Clover lig.
4 white
1 blue (control)
The next plasmids were prep to be used in the formation of L2 ligation.
-PAGM8031
-PlCH50900
Spec results after mini prep (Concentration record in ng/ul)
White 1-245
White 2-262.5
White 3-255.7
White 4-291.6
Blue-111.5
PAGM8031-102.2
PICH50900-28.3

September 11, 2023

L1 PSR 1 Sma I diagnostic digestion

September 11, 2023

The following reaction was performed for the 4 colonies pick plus a control.
Plasmid DNA: 4uL
buffer: 1uL
Sma1: 1uL
H2O: 4uL
total: 10uL
Incubate for 1 hour at 37°C

September 12, 2023

L1 Diagnostic Digest of L1-Psr-HA-Clover colonies

September 12, 2023

A diagnostic digest was performed for the L1-Psr-HA-Clover colonies with Noel and Nde:
Plasmid DNA: 7uL
Cutsmart buffer: 1uL
Not1: 1uL
Nde: 1uL
total: 10uL
Incubate for 1 hour at 37°C

Key: Ladder; 2+4 showed expected lines at 4.5, 3.5, 1.5kb; 3+5 showed extra lines, 6 negative control.
This data supports the correct assembly of L1 plasmid of clones present in lanes 2 and 4.
-Clone present on line 2 was used to proceed with the formation of L2 plasmid.

September 18, 2023

PSR1 L2 Plasmid Ligation

September 18, 2023

For L2 ligation next protocol was followed:
Plasmid(Psr1 L1): 1uL
PAGM8031: 1uL
TCrnpt: 0.9uL
H2O: 23.2uL
54022: 0.9uL
50892: 0.6uL
This was then split into two tubes, making 14 ul in each. See the excel sheet below for the rest of the information.

High Efficiency Transformation was performed with the L2 PSR1 Ligation, using NEB 10-beta competent E. coli cells.

September 20, 2023

L2 PSR1 Liquid culture inoculation

September 20, 2023

20 Colonies from the transformed cells plated were picked to be inoculated in a liquid culture.
Pipette tips were used to streak the plate, then put into corresponding labeled tubes. They were placed in the shaking incubator at 37°C to grow.

September 21, 2023

L2 PSRS1 Plasmid mini prep/digest

September 21, 2023

The 20 preps from yesterday were mini prepped:

They were then digested as follows:
Master mix:
ddH2O: 300uL
CS buffer: 46uL
EcoR1: 23uL

Each tube:
MM: 16uL
plasmid: 4uL
total: 20uL

Incubate at 37°C for 1 hr

All the colonies with the L2 PSR1 Plasmid showed the expected fragments at 9.9 and 2.2 Kb, while the ones coming from the blue colonies (empty vector controls) show the expected different pattern on the band size. Showing that the L2 PSR1 plasmid was constructed correctly.

August 31-September 27, 2023

Failed attempts at L2-nitrogen cloning

August 31-September 27, 2023

Repeated attempts at building the L2-nitrogen plasmid that would express aadA for specR, HAO, AMO, and nosZ failed. After troubleshooting we determined that the L1-HAO plasmid was incorrect due to an incorrect interpretation of our diagnostic digest on August 1, causing our Level 2 assembly to fail.

September 27, 2023

L2 PSR1 Plasmid electroporation into Chlamydomonas reinhardtii

September 27, 2023

Plasmid electroporation protocol was performed to incorporate the L2 plasmid construct into Chlamydomonas reinhardtii.

September 28, 2023

C. reinhardtii plating

September 28, 2023

Electroporated cells were plated on TAP + kan plates.

September 29, 2023

Golden Gate Assembly of L1-HAO

September 29, 2023

We set up another ligation reaction of the L1-HAO plasmid, and transformed it into NEB10beta cells.

September 30, 2023

L1 HAO ligations continued

September 30, 2023

We inoculated liquid cultures with colonies from the plates. Colonies 1 and 2 were from the 10 beta plates, colonies 3-12 were from the stable plates. 1-11 were white colonies, 12 was blue. Also prepared a streak plate with streaks from each colony. All were incubated at 30 C starting at 11am.

October 1, 2023

L1 HAO miniprep, HindIII digest, and sequencing

October 1, 2023

Miniprepped liquid cultures from yesterday and eluted with 30 uL elution buffer
For HindIII digest, mixed 3 uL of plasmid with 14 ul ddH20, 2 uL CS buffer and 1 uL HindIII. Also digested L1 HAO Flag #2 plasmid that turned out to be an incorrect assembly. Correct assembly should produce 5 kb and 2.7kb fragments.
For sequencing reactions, mixed 3 uL of plasmid with 6 uL ddH20 and 1 uL of 25 uM RB-F1 sequencing primer

Plasmid ng/uL A260/270 260/230
L1 HAO 10 beta 1 140 1.85 1.84
L1 HAO 10 beta 2 -2 N/A N/A
L1 HAO 10 stable 3 206 1.7 1.0
L1 HAO 10 stable 4 188 1.86 1.99
L1 HAO 10 stable 5 215 1.86 1.96
L1 HAO 10 stable 6 247.5 1.85 1.75
L1 HAO 10 stable 7 152 1.7 1.1
L1 HAO 10 stable 8 168.8 1.87 2.0
L1 HAO 10 stable 9 145.1 1.81 1.5
L1 HAO 10 stable 10 235 1.88 2.1
L1 HAO 10 stable 11 99.4 1.88 2.14
L1 HAO 10 stable 12(blue) 174 1.8 1.4


The lanes marked with an asterisk are digests with the expected fragment size. Subsequent sequencing of clone 4 supports successful build of the L1-HAO-Flag plasmid
Set up a L2 ligation reaction using clone #4, since it had a good yield via spec and promising digest results.

October 2, 2023

Transformation of L2-Nitrogen Ligations

October 2, 2023

Transformed into NEBStable cells using standard heat-shock protocol.

October 3, 2023

Inoculation of liquid cultures

October 3, 2023

Inoculated LB+spec broth with the colonies, incubated at 30C overnight.

October 4, 2023

Miniprep and diagnostic digest of L2-Nitrogen plasmids

October 4, 2023

Miniprepped the liquid cultures:

Plasmid Concentration(ng/uL)
1 111.4
2 98
3 108.3
4 121
5 93.4
6 113.7
7 193
9 10.4
10 121.1
11 61.3
12 102.8
blue 99.5

Set up diagnostic digests of the plasmids with HindIII
Reagent 1rxn(uL) 16 rxn(uL)
ddH2O 12 198
Cutsmart buffer 2 32
HindIII 1 16
DNA 5 -

Samples incubated at 37C for an hour. Also digested one of the colonies from 9/21.
Expected fragment sizes are 7.6, 6.7, and 2.7 kb

Mostly looks good, except that the miniprep marked "blue" produces a pattern expected for the L2-nitrogen plasmid: 7.6, 6.4, and 2.7 kb. #9 looks like the empty vector. Subsequent discussion suggested that colonies #9 and blue were switched during the miniprep process.
In conclusion, the diagnostic digest supports the successful creation of the L2-Nitrogen plasmid in minipreps 1,2,4,5,6,7,10,11, and 12.

October 4, 2023 part 2

Linearization of Plasmid for electroporation into Chlamydomonas reinhardtii

October 4, 2023

1 ug of L2-Nitrogen clone #2 was digested with BsaI in a 10 uL reaction at 37C for one hour, then heat-killed at 80C for 20 min. This reaction was used to as the DNA for electroporation.

October 5, 2023

Growth

October 5, 2023

Successful growth of transformed colonies observed, showing expression of the plasmid via antibiotic resistance.

October 5, 2023

Electroporation of L2-Nitrogen plasmid into Chlamydomonas reinhardtii

October 5, 2023

We electroporated using the Electroporation Protocol.

October 6, 2023

Success!

October 6, 2023

After a week of growth cell were checked for expression of fluorescence.


The presence of fluorescence shows the successful electroporation of the L2 PSR1 Plasmid into the Chlamydomonas reinhardtii cells, and not only that, but that our Psr1 clover construct is being expressed, not just the antibiotic resistance.

October 11, 2023

Success!

October 11, 2023

We observed spec-resistant colonies of C. reinhardtii growing 6 days after electroporation, suggesting successful integration of the L2-nitrogen construct.

Later the same day we confirmed that our C. reinhardtii are expressing our L2 nitrogen construct as seen by the fluorescent tag!


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