Week 1
Week 1 was spent developing our initial approach and wetlab design.
Week 2
Thursday 6/12/2025
Selected TSS method for preparing competent cells
Prepared buffers for TSS method
Week 3
Sunday 6/15/2025
Prepped 3 overnight cultures of E. coli BW25113 (BW25113)
Monday 6/16/2025
Inoculated E. coli BW25113 into 1200 mL LB medium
Original plate with the E. coli BW25113 was re-streaked
Poured plates with kanamycin antibiotic
Tuesday 6/17/2025
SM-1 media fell out of solution
Prepared TSS buffer
Made glycerol stocks of E. coli BW25113
Wednesday 6/18/2025
Organized lab setup and labeled primary workstations.
Cleaned countertops and lab equipment
Ordered remaining reagents for SM-1 Media
Performed a transformation of E. coli BW25113 using the TSS method
Thursday 6/19/2025
Analyzed results from 6/18 Transformation
Performed a transformation of E. coli BW25113 using the TSS method with different ampicillin plates
Friday 6/20/2025
Analyzed results from 6/19 transformation.
Obtained new plates of E. coli MG1655 and E. coli JM109.
Re-streaked colonies of E. coli BW25113, E. coli MG1655 and E. coli JM109
Saturday 6/21/2025
Analyzed re-streak of E. coli BW25113, E. coli MG1655 and E. coli JM109
Concluded that the E. coli BW25113 was not E. coli BW25113
Week 4
Monday 6/23/2025
Sample transformation with E. coli JM109
Tuesday 6/24/2025
Analyzed results from 6/23 transformation
Transformed L-cysteine overexpression plasmid from Twist Bioscience (TV1) into E. coli JM109
Wednesday 6/25/2025
Analyzed results from 6/24 transformation.
PCR of L-cysteine overexpression plasmid.
Ran gel electrophoresis for the PCR product.
Colonies from 6/24 transformation were re-streaked on to new ampicillin plates.
Thursday 6/26/2025
Analyzed 6/25 re-streaked plates.
Prepared large volume of SM-1 media.
Transformation of TV1 into E. coli JM109
Friday 6/27/2025
Analyzed results from 6/26 transformation.
Week 5
Sunday 6/29/2025
Prepared overnight cultures of transformed E. coli JM109 containing TV1.
Monday 6/30/2025
Mini-prepped the E. coli JM109 from 6/29 overnight cultures.
Ran a restriction digest of TV1 with PstI and XbaI.
Prepared and froze stocks of from 6/29 overnight culture.
Ran a ninhydrin calibration curve.
Prepared 100X stock solution of L-cysteine-hydrochloride.
Thursday 7/3/2025
Additional 100X L-cysteine-hydrochloride stock prepared.
Prepared overnight cultures of transformed E. coli JM109 containing pUC19 and TV1.
Week 6
Sunday 7/6/2025
Transformed E. coli JM109 colonies with pUC19 and TV1.
Monday 7/7/2025
Checked on the 7/6 transformed E. coli JM109 cultures
Inoculated the transformed E. coli JM109 cultures into SM-1 media, with OD600 readings taken every hour.
Ran ninhydrin assay with calibration curve
Obtained a plate of E. coli BW25113 and re-streaked colonies onto a new plate.
Tuesday 7/8/2025
Inoculated E. coli JM109 containing pUC19 and TV1 into SM-1 media.
Prepared fresh ninhydrin reagent 2.
Analyzed re-streaked 7/7 E. coli BW25113 and prepared overnight cultures.
Wednesday 7/9/2025
Prepared fresh ninhydrin reagent 2
Mini-prepped 7/8 E. coli JM109 and sent for DNA sequencing.
Ran a restriction digest from mini-prepped DNA.
Ran a ninhydrin assay calibration curve.
Prepared fresh L-cysteine-hydrochloride stock solution.
Thursday 7/10/2025
Prepared fresh ninhydrin reagent 2
Ran ninhydrin assay
Changes to calibration curve methodology; made L-cysteine-SM-1 stock.
Friday 7/11/2025
Observed that 7/10 L-cysteine-SM-1 stock had fallen out of solution.
Made additional changes to calibration curve methodology, switching back to L-cysteine-hydrochloride stocks. Performed a calibration curve with the changes.
Performed various tests to refine the steps of the ninhydrin assay.
Analyzed sequencing data from Azenta.
Transformed TV1 and pUC19 into E. coli DH5α cells.
Saturday 7/12/2025
Analyzed results from 7/11 transformation.
Prepared overnight cultures from 7/11 transformation product.
Week 7
Sunday 7/13/2025
Glycerol stocks of E. coli DH5α containing TV1 from 7/11 overnight culture were prepared and stored in -80 °C freezer
Mini-prepped E. coli DH5α containing TV1 from 7/11 overnight culture.
Mini-prepped DNA was transformed into E. coli JM109 cells.
Ran more tests to optimize ninhydrin assay calibration curve to understand impact of each step.
Monday 7/14/2025
Restriction digest of 7/13 mini-prepped TV1 from DH5α cells
Second digest was run under the same conditions
Analyzed 7/13 E. coli JM109 transformation product
Prepared overnight cultures of pUC19 and TV1 from 7/13 E. coli JM109 transformation product
Tuesday 7/15
Overnight colonies from 7/14 were inoculated into SM-1 media
Prepared fresh ninhydrin reagent 2
Ran ninhydrin assay
Ran ninhydrin assay calibration curve
Mini-prepped overnight colonies from 7/14
Wednesday 7/16
Inoculated overnight cultures into SM-1 media
Prepared fresh ninhydrin reagent 2
Ran ninhydrin assay calibration curve
Thursday 7/17
TV1 DNA from E. coli DH5α (7/13) and E. coli JM109 (7/15) sent for Sanger sequencing
Prepared overnight cultures of E. coli JM109.
Friday 7/18
Performed linearization of pUC19 vector via restriction digest at XbaI site
Transformed TV1 into NEB® Turbo Competent E. coli (High Efficiency) (Turbo)
Saturday 7/19
Analyzed results from 7/18 transformation.
Prepped 8 overnights from the transformed E. coli DH5α plate of TV1
PCR and gel electrophoresis imaging of L-cysteine triggered kill switch plasmid from IDT (Insert 2)
Prepared overnight cultures of Turbo E. coli containing TV1.
Week 8
Sunday 7/20
Mini-prepped overnight cultures of Turbo E. coli
Purified 7/19 Insert 2 PCR product
Created a stock of competent E. coli MG1655 using the TSS method for competent cells. The stocks were stored in an –80oC freezer.
Transformed E. coli JM109 with TV1 from 7/20 miniprep.
Stocks of DH5α were prepared and stored.
Transformed Stellar competent E. coli cells with In-Fusion Insert 2 product.
Monday 7/21
Analyzed results from 7/20 E. coli JM109 TV1 transformation.
Analyzed results from 7/20 Stellar cells Insert 2 transformation
TV1 in Turbo cells (7/20) sent for sequencing through Azenta
Performed restriction digest of TV1 in Turbo cells with BbsI.
Tuesday 7/22
Performed In-Fusion assembly for Insert 2
Ran test live/dead assay for Insert 2
Prepared overnight cultures of Stellar cells containing Insert 2
Wednesday 7/23
Performed In-Fusion assembly for Insert 2
Ran live/dead assay for Insert 2
Collected cell lysates and started Western Blot
Transformation of TV1 into Turbo cells
Transformation of Insert 2 into Stellar cells
Thursday 7/24
Analyzed results from 7/23 Western Blot
Analyzed results from 7/23 TV1 transformation into Turbo cells.
Analyzed results from 7/23 Insert 2 transformation into Stellar cells.
Saturday 7/26
Overnight cultures prepared from 7/23 transformation products of both TV1 and Insert 2
Week 9
Sunday 7/27
Mini-prepped both TV1 and Insert 2 from 7/26 overnight cultures.
Transformed mini-prepped TV1 into E. coli JM109.
Transformed mini-prepped Insert 2 into E. coli MG1655.
Monday 7/28
Analyzed both 7/27 transformation results
Performed restriction digest of TV1 using BbsI
Performed restriction digest of Insert 2 using XbaI
Performed colony PCR on E. coli JM109 transformed with TV1.
Resuspended and transformed Anderson promoters from the iGEM 2025 Distribution Kit into E. coli DH5α.
Sent TV1 and Insert 2 DNA from 7/27 mini-prep for sequencing.
Prepared overnight cultures of E. coli MG1655 and Stellar cells, both transformed with Insert 2
Tuesday 7/29
Ran colony PCR products via gel electrophoresis.
Created a new method for validating Insert 2.
Ran cell viability assay and optical density readings with Stellar cells containing Insert 2.
Transformed resuspended Anderson Promoters J23102, J23105, J23109 from the iGEM 2025 Distribution Kit into Turbo cells
Wednesday 7/30
Officially confirmed via sequencing results that the DNA sent to us by Twist Bioscience was incorrect, as it contained no homology to our intended insert.
Prepared overnight cultures of E. coli MG1655 containing Insert 2.
Prepared competent E. coli MG1655 cells using the TSS method.
Thursday 7/31
Mini-prepped from 7/30 overnight E. coli MG1655 Insert 2 culture.
Digested mini-prepped DNA with XbaI.
Sent mini-prepped Insert 2 for sequencing.
Test transformation of E. coli MG1655 competent cells from 7/30 with pUC19.
Prepared overnight cultures of Stellar cells transformed with Insert 2.
Prepared overnight cultures of Turbo cells transformed with Anderson promoters.
Friday 8/1
Mini-prepped from 7/31 overnight Stellar cell Insert 2 cultures
Digested mini-prepped DNA with XbaI
Sent mini-prepped Insert 2 for sequencing.
Transformed DNA from selected colonies into E. coli MG1655 based on digest results.
Saturday 8/2
Analyzed results from 8/1 transformation
Prepared overnight cultures of E. coli MG1655 from 8/1 transformation.
Week 10
Sunday 8/3
Insert 2 Assay including cell viability and optical density readings from 8/2 E. coli MG1655 overnight cultures was performed.
Prepared overnight cultures of E. coli MG1655 from 8/1 transformation.
Monday 8/4
Ran Insert 2 bacterial viability assay
Performed In-Fusion assembly for Insert 2
Prepared overnight cultures of E. coli containing Insert 2
Tuesday 8/5
Transformed 8/4 In-Fusion product into Turbo competent cells.
Performed bacterial viability assay calibration curve.
Prepared overnight cultures of Turbo cells containing Insert 2
Wednesday 8/6
Mini-prepped DNA from 8/5 overnight cultures containing Insert 2.
Performed restriction digest on mini-prepped Insert 2 DNA using XbaI.
Sent mini-prepped DNA for sequencing based on digest results.
Week 12
Monday 8/18
Received E. coli CcdB survival cells (CcdB survival).
Performed restriction digest of the L-cysteine overexpression plasmid from ANSA Biotechnologies (Insert 1).
PCR of Insert 1 and linearized pACYC to add homologous ends.
Tuesday 8/19
Performed restriction digest of Insert 2 DNA from 8/1 and 8/6 mini-preps using BbsI and BtgI.
Performed ligation of Insert 2 using 8/19 digest product.
Transformed ligation product into CcdB survival cells.
Wednesday 8/20
pACYC from GoldBio was transformed into Turbo competent cells.
Analyzed results from 8/19 CcdB survival cell transformation with Insert 2 ligation product.
Prepared overnight cultures from 8/19 CcdB survival cell transformation with Insert 2 ligation product.
Thursday 8/21
Analyzed results from 8/20 Turbo cell transformation with pACYC.
Mini-prepped 8/20 CcdB survival overnight cultures containing Insert 2.
Performed restriction digest of mini-prepped Insert 2 DNA using BbsI and BtgI.
Sent mini-prepped Insert 2 DNA for sequencing based on digest results.
Prepared overnight cultures of 8/20 Turbo cell transformation with pACYC.
Friday 8/22
Mini-prepped 8/21 Turbo cell overnight cultures containing pACYC.
Performed PCR of Insert 1.
Prepared overnight cultures of 8/20 Turbo cell transformation with pACYC.
Analyzed sequence results from 8/21 mini-prep.
Saturday 8/23
Mini-prepped 8/22 Turbo cell overnight cultures containing pACYC.
Performed restriction digest of mini-prepped pACYC DNA using XbaI.
Performed restriction digest of Insert 2 from 8/1 and 8/6 mini-preps using BbsI and BtgI.
Week 13
Wednesday 8/27
Performed restriction digest of Insert 2 DNA from 8/1 and 8/6 mini-preps using BbsI and BtgI.
Performed gel extraction of restriction digest product.
Performed ligation of Insert 2 using 8/27 digest product.
Transformed ligation product into CcdB survival cells.
Thursday 8/28
Analyzed results from 8/27 transformation.
Friday 8/29
Performed restriction digest of Insert 2 DNA from 8/1 and 8/20 mini-preps using BbsI and BtgI.
Performed ligation of Insert 2 using 8/29 digest product.
Performed In-Fusion assembly of Insert 1.
Saturday 8/30
Transformed 8/29 In-Fusion product into Stellar cells.
Transformed 8/29 ligation product into CcdB survival cells.
Week 14
Sunday 8/31
Analyzed results from 8/30 transformations.
Monday 9/1
Secondary analysis of results from 8/30 transformations.
Transformed 8/29 In-Fusion product into Stellar cells.
Transformed 8/29 ligation product into CcdB survival cells.
Tuesday 9/2
Analyzed results from 9/1 transformations.
Transformed 8/29 In-Fusion product into Stellar cells.
Prepared overnight cultures of 9/2 Stellar cell transformation with Insert 1 and pACYC.
Wednesday 9/3
Mini-prepped 9/2 Stellar cell overnight cultures containing Insert 1 and pACYC.
Performed restriction digest of Insert 1 and pACYC DNA from 9/3 mini-prep using EcoRI-HF.
Sent mini-prepped Insert 1 DNA for sequencing based on digest results.
Thursday 9/4
Prepared overnight cultures of 9/2 Stellar cell transformation with Insert 1 and pACYC.
Friday 9/5
Mini-prepped 9/4 Stellar cell overnight cultures containing Insert 1 and pACYC.
Performed PCR for both Insert 1 in pIND backbone and Insert 2.
Prepared overnight cultures of pACYC (in turbo cells, 8/20) and pUC19 (in CcdB survival, 8/19) for use in digest experiment and vector linearization.
Saturday 9/6
Mini-prepped overnights cultures of CcdB survival cells and Turbo cells with empty vector.
Performed restriction digest of pUC19 using XbaI.
Performed restriction digest of pACYC using EcoRI.
Week 15
Sunday 9/7
Ran products from 9/6 digests via gel electrophoresis.
Ran products of 9/5 PCR of Insert I and Insert 2 via gel electrophoresis and analyzed results.
Performed PCR for both Insert 1 in pIND backbone and Insert 2.
Monday 9/8
Ran products of 9/7 PCR of Insert I and Insert 2 via gel electrophoresis and analyzed results.
Wednesday 9/10
Performed PCR of Insert 1 in pIND backbone.
Performed restriction digest of pUC19 and pACYC using XbaI.
Performed second restriction digest of pUC19 and pACYC using XbaI and fresh nuclease-free water.
Performed gel extraction of PCR product and pUC19& pACYC digest product.
Performed In-Fusion assembly of Insert 1 and Insert 2
Transformed Insert 1 into Stellar and Insert 2 into CcdB survival cells using 9/10 In-Fusion products.
Thursday 9/11
Analyzed results from 9/10 transformations.
Saturday 9/13
Secondary analysis of 9/10 transformation product.
Transformed Insert 1 into Stellar and Insert 2 into CcdB survival cells using 9/10 In-Fusion products.
Week 16
Sunday 9/14
Analyzed results from 9/13 transformations.
Monday 9/15
Performed colony PCR of In-Fusion transformed plates.
Tuesday 9/16
Prepared overnight culture of Stellar cells containing Insert 1.
Wednesday 9/17
Mini-prepped 9/16 Stellar cell overnight cultures containing Insert 1.
Performed restriction digest of pUC19 and pACYC using XbaI and Calf Intestinal Phosphatase (CIP).
Thursday 9/18
Performed gel extraction for pUC19 and pACYC 9/17 digest product.
Week 17
Sunday 9/21
Transformation of linearized pUC19 and pACYC into Stellar cells using 9/18 gel extraction product.
Monday 9/22
Analyzed results from 9/21 transformation.
Performed In-Fusion assembly of Insert 1 and Insert 2.
Transformed Insert 1 into Stellar and Insert 2 into CcdB survival cells using 9/22 In-Fusion products.
Transformed linearized pUC19 and pACYC into Stellar cells
Tuesday 9/23
Analyzed results from 9/22 transformation.
Prepared overnight cultures of 9/22 Stellar cell transformation with Insert 1.
Wednesday 9/24
Mini-prepped 9/23 Stellar cell overnight cultures containing Insert 1.
Performed digest of Insert 1 using XbaI.
Transformed Insert 2 into CcdB survival cells.
Thursday 9/25
Prepared overnight cultures of 9/24 CcdB survival cell transformation with Insert 2, Insert 2 negative control and Insert 1 negative control.
Friday 9/26
Mini-prepped 9/25 overnight cultures.
Performed digest of Insert 2 DNA using BbsI and BtgI.
Week 18
Sunday 9/28
Transformed Insert 1 DNA from ANSA into Turbo cells.
Monday 9/29
Re-streaked 9/28 transformation product onto a new plate.
Tuesday 9/30
Prepared overnight cultures from 9/29 re-streaked plate of Turbo cells containing Insert 1.
Wednesday 10/1
Cloned Insert 1 and Insert 2 into pUC19 via traditional restriction cloning to reduce background and utilize blue-white colony screening.
Mini-prepped 9/30 overnight cultures.