Team:ASU/Notebook
Project Notebook
A comprehensive record of all of the procedures and experiments conducted over the course of the project.
Lab Work
×JUNE 24
- Midiprep done to obtain the backbone plasmid, pASapI.JUNE 25
- Verified pASapI with nanodrop and restriction digest.JUNE 28
- Miniprepped metallothionein (MT). Extracted DNA from transformed colonies, and confirmed that insert was present through nanodrop and restriction digest.JUNE 29
- Gel extraction of MT plasmid, as well as Golden Gate of asrC and ferritin A (ftnA).JULY 2-3:
- Miniprep, nanodrop, and restriction digest verification of arsC and ftnA.JULY 5:
- Ran Golden Gate assembly of tagged MT, ftnA, and arsC.JULY 6:
- Transformed tagged sequences.- Completed first trial run of shooting with gene gun. Control round completed with backbone pASapI with no inserts.
JULY 7:
- Retransformation of tagged sequences after previous attempt failed.JULY 9:
- Miniprep and restriction digest to verify transformation of tagged genes.JULY 14:
- First trial run of shooting C. reinhardtii with gene gun.- Round 1 involved MT, arsC, ftnA, as well as tagged MT, tagged arsC, and tagged ftnA.
JULY 19-21:
- Attempted Golden Gate assembly of recombinated DNA, but proved to be unsuccessful.- Transformations did not result in enough colonies on the positive plate.
JULY 22:
- Colony PCR attempted, but was unsuccessful because primers were not sensitive enough for colony PCR.JULY 26:
- Colony PCR attempt 2, which also produced no viable results.JULY 29-30:
- Completed Golden Brain assembly of asrC-ftnA.- Troubleshot issues in cloning by switching to IEE5 (required ordering new constructs with IEE5).
June + July 2021
Lab procedures and experiments from June/July 2021.
Lab Work
×AUGUST 7-8:
- Completed Golden Gate transformation with new IEE5 sequences.- Success with MT-ftnA and MT-arsC-ftnA sequences.
AUGUST 9:
- Miniprep and restriction digest verification of new sequences.AUGUST 13:
- Second attempt of combining sequences, using regular T4 in place of the high-fidelity T4 that was being used before.- Two more successes in combining Mt-arsC and MT-ftnA.
AUGUST 17:
- Miniprep, nanodrop, and restriction digest verification of MT-arsC and MT-ftnA sequences.AUGUST 20:
- Second session of shooting C. reinhardtii with gene gun, working with pASapI, arsC, ftnA, MT, as well as tagged arsC, tagged ftnA, and tagged MT.AUGUST 25:
- Golden Braid of both full and tagged sequences: arsC, ftnA, and MT, as well as tagged arsC, tagged ftnA, and tagged MT.- Ordered new enzymes for use in lab, and realized that restriction digest verification was not actually indicative of success in transformation.
- Stopped working with full sequences, as there was not enough time to shoot them into C. reinhardtii.
AUGUST 26-31:
- Miniprep, nanodrop, and restriction digest verification of remaining sequences.August 2021
Lab procedures and experiments from August 2021.
Lab Work
×SEPTEMBER 1
- Transformed normal, tagged, and pASapI DNA sequences into E. coli.SEPTEMBER 2
- Big miniprep session for glass bead transformations of the following: arsC, ftnA, MT, as well as tagged arsC, tagged ftnA, and tagged MT.SEPTEMBER 3
- Continued the glass bead transformations.SEPTEMBER 9-14
- Trial run of arsenic: wildtype algae in culture with a sample of Arsenic in the following quanitites: 50 ppb, 500 ppb, and 5ppm.- Collected samples at 24 hours, 48 hours, and 72 hours, and stored the samples to run further analyses on.
SEPTEMBER 16
- Ran ICP-MS analyses on samples.- Diluted samples with nitric acid, but experienced a machine malfunction.
SEPTEMBER 17
- Noticed 3 colonies on plates from August:1. MT plate 4 from 20 August (struck onto new plate, labeled iGEM C1).
2. arsC HA plate 4 from 20 August (struck onto new plate, labeled iGEM C2).
3. MT 6XHis plate 4 from 20 August (struck onto new plate, labeled iGEM C3).
SEPTEMBER 20-21
- Re-prepped ICP-MS analyses (run at Westerhoff Lab).SEPTEMBER 27
- Received results of ICP-MS analyses.- Moved samples iGEM C1 and iGEM C2 from Redding Lab to Bartelle Lab.
SEPTEMBER 30 - OCTOBER 3
- Started trials on iGEM C1 and arsenic (As), using three biological replicates of C. reinhardtii in 50 ppb and 500 ppb.- Samples collected at 48 and 72 hours.
September 2021
Lab procedures and experiments from September 2021.
Lab Work
×OCTOBER 2
- 48-hour sample of sample C1 collected.OCTOBER 3
- 72-hour sample collection of sample C1.- Began new C2 culture.
OCTOBER 7
- Began As trials on sample C2, and 0-hour time sample collected.OCTOBER 9
- Collected 48-hour samples of C2.OCTOBER 10
- Collected 72-hour sample of C2, and began C3 trial (collected 0-hour sample).OCTOBER 12
- Collected 48-hour sample of C3.OCTOBER 13
- Collected 72-hour sample of C3.- Began wildtype-As trials, and collected 0-hour sample.
OCTOBER 15
- Collected 48-hour sample of wildtype-As trial.- Prepped C2, C3, and wildtype samples for western blot.
- Acidified samples for ICP-MS analyses in Westerhoff Lab.
OCTOBER 16
- Collected 72-hour analyses of wildtype-As trial samples.OCTOBER 18
- Conducted western blot of C2, C3, and wildtype samples.- Acidified 48-hour and 72-hour samples from October 16th.
- Ran ICP-MS analyses via Westerhoff Lab.
OCTOBER 19
- Completed imaging of the western blot.October 2021
Lab procedures and experiments from October 2021.