Notebook

July

July 23

Prepared LB Agar Plate and Broth containing appropriate antibiotics for the Interlab study.

July 24

Started InterLab Study 1 and 3 Day 0 protocol
Transformation protocol used:
  1. Obtain agar plates and label them appropriately.
  2. Aliquot 25 uL of competent E. coli DH5-alpha into an Eppendorf tube (Initial volume in each tube contains 50).
  3. Add 2 uL of plasmid into the first tube containing the competent E. coli. Allow the stored resuspended distribution kit to thaw before use. Store kit back at -20°C.
  4. Place mixture on ice for 20 minutes.
  5. Heat-shock the bacteria for 60 seconds using a heat block at 42°C.
  6. Immediately place tubes on ice for 2 minutes.
  7. Add 970 uL of SOC.
  8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
  9. Spread gently using a plastic spreader.
  10. Incubate overnight (16 hours) at 37°C.

July 25

Transformations were unsuccessful. Possibly because the final solution plated had too much SOC and, upon filling the plate, the solution flowed off the plate.

July 26 – August 29: Summer break

August

August 30

  1. Weighed out 8.75 g of LB Agar and 10 g of LB Broth and added it to 250 mL and 500 mL of diH2O respectively.
  2. Sealed the flask with aluminum foil and put autoclave tape on top.
  3. Placed both flasks in the autoclave. Ensured the water level inside the autoclave was sufficient and the water level was below the low marking on the water tank at the bottom right.
  4. Started the autoclave using Mode 4. Flasks can be left in the autoclave overnight because of the keep warm feature (50°C).

August 31

Preparation of Agar Plates and Broth
  1. 250 uL of antibiotic was added to the pre-prepared LB Agar.
  2. Added appropriate antibiotic to the LB Agar solution.
  3. Prepared and filled 20 Petri dishes with the LB Agar.
  4. Petri dishes were left to dry.
  5. LB Broth that was left overnight was foamy, and signs of contamination were detected, so a new broth was made.
Transformation by Heat Shock
  1. Obtain agar plates and label them appropriately.
  2. Aliquot 25 uL of competent E. coli DH5-alpha into an Eppendorf tube (Initial volume in each tube contains 50).
  3. Add 2 uL of plasmid into the first tube containing the competent E. coli. Allow the stored resuspended distribution kit to thaw before use. Store the kit back at -20°C.
  4. Place mixture on ice for 20 minutes.
  5. Heat-shock the bacteria for 60 seconds using a heat block at 42°C.
  6. Immediately place tubes on ice for 2 minutes.
  7. Add 970 uL of SOC.
  8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
  9. Centrifuge for 2 minutes at 13,000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
  10. Carefully resuspend the pellet by pipetting up and down and transferring 50uL of transformed bacteria to the respective agar plate containing the appropriate antibiotic.
  11. Spread gently using a plastic spreader.
  12. Incubate overnight (16 hours) at 37°C.

September

September 1

  1. Checked on the transformation to verify successful transformation. 3 plates did not have any growth on them.
  2. LB Broth was contaminated again. The broth (500 mL) was prepared again using a new LB Broth powder.

September 4

  1. Received training for Biotek Cytation 5 microplate reader and Excella Shaker with Sneha Ann Thomas, CTP Molecular and Cell Biology
  2. Performed calibration portion of the InterLab Study.

September 5

  1. Repeated transformations for Interlab study for wells 13A, 13C, 13G.
  2. Inoculated the transformations that worked into liquid broth following the InterLab protocol.

September 6

  1. Transformations still didn’t work for Interlab. Growth was observed on the positive controls. Constructs in the well could have been degraded because of storage over the break.

September 7

  1. Prepared agar plates with kanamycin
Resuspension of lyophilized plasmid
  1. Keep the vial sealed and on ice until ready to use.
  2. Centrifuge at 6,000x g for 1 minute at 4°C.
  3. Resuspend pQE-80L-Kan in 10 uL of IDTE.
  4. Resuspend pUC57-Kan containing collagen-like region from Scl2(CO) in 20 uL of nuclease-free water.
  5. Keep both tubes on ice and proceed with the transformation of competent DH5 alpha cells.
Streak E. coli Auxotroph strain

E. coli Keio Knockout Strain (glycerol stock) JW id: JW0233 Strain: 2 ECK number: ECK0244 Escherichia coli MG1655 B id: b0243 Gene name: proA

E. coli Keio Knockout Strain (glycerol stock) JW id: JW2806 Strain: 1 ECK number: ECK2836 Escherichia coli MG1655 B id: b2838 Gene name: lysA

September 8

  1. Transformation from yesterday did not work.
  2. Repeated transformations for pQE-80L-Kan and pUC57-Kan containing the collagen-like region from Scl2(CO). Positive control was included to check if the error was in the protocol.

September 10

  1. Both transformations worked.
  2. Multiple individual colonies were picked and inoculated into individual tubes containing 6 mL of LB Broth with Kan.

September 11

  1. Performed MiniPrep for pQE80L-Kan and pUC57-Scl2.28 following the standard protocol listed on the experiments page.
  2. Performed Nanodrop measurements for the samples. The yield of the plasmid in (ng/uL) was noted on the tubes. 260/280 were in the appropriate range.

September 12

  1. Prepared 1% agarose gel.
  2. Double digest of pQE80L-Kan and pUC58-Scl2.28 using BamHI-HF and HindIII-HF for 25 minutes.
  3. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest, pUC57- Kan with Scl2.28, pUC57-Kan with Scl2.28 double digest using appropriate ladder.
  4. Successful double digestion of pQE80L-Kan could not be determined. More details on engineering success page.

September 13

  1. Prepared 1% agarose gel.
  2. Double digest of pQE80L-Kan was repeated following the same procedure but for a different tube of Miniprepped plasmid.
  3. pUC57-Scl2.28 using single digested HindIII-HF to determine size of linearized plasmid.
  4. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest, pQE80L-Kan Sample 2, pQE80L-Kan Sample 2 double digest, pUC57-Kan with Scl2.28 double digest, and pUC57-Kan with Scl2.28 single digest.

September 14

  1. Prepared 3% agarose gel.
  2. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest, and pQE80L-Kan double digest S2 to check for fragments between the restriction sites.
  3. Double digestion of pQE80L with BamHI and HindIII for 1 hour and 2 hours. Digestion was also performed sequentially BamHI followed by HindIII for 30 minutes each.
  4. Gel electrophoresis was performed for pQE80L-Kan, pQE80L-Kan double digest 1hr, pQE80L-Kan double digest 2hr, and pQE80L-Kan single digest.

September 15

  1. Single colonies of pQE80L in DH5-alpha were incubated in 5 mL of LB Broth.

September 21

  1. Plasmid miniprep of overnight culture of pQE80L.
  2. Nanodrop of extracted plasmid.
  3. Two different samples of double digested pQE80L were ligated with Scl2.28 using T7 ligase.

September 22

  1. E. coli BL21 was transformed using ligated product.

September 23

  1. Plasmid was extracted from overnight liquid culture through Miniprep.
  2. Extracted plasmid purity was measured using Nanodrop.
  3. Extracted plasmid was double digested using BamHI and HindIII to verify the correct sequence.
  4. Gel electrophoresis of extracted plasmid and double digested product.

September 24

  1. pQE80L double digested for 30 min, 1 hour, and 2 hour was ligated to double digested pUC57-Kan with Scl2.28 using T7 ligase.
  2. Each plasmid was used to transform BL21.

September 25

  1. Miniprep of pQE80L-Kan overnight liquid culture.
  2. The purity of extracted plasmid was quantified using Nanodrop.

September 26

  1. Single colonies were observed on all three combinations.
  2. pQE80L digested for 20 mins using BamHI followed by 20 mins using HindIII.
  3. Ligation of the two successive single digested pQE80L and double digested pQE80L with Scl2 was performed using T4 Ligase.
  4. Transformation into DH5-alpha.

September 27

  1. Prepared 500 mL of LB Broth with Kan.
  2. Inoculated single colonies of each construct into LB Broth..

September 28

  1. Miniprep of liquid culture was performed using standard procedure.
  2. Nanodrop was used to quantify the purity of plasmid DNA.
  3. PCR of plasmid to verify successful ligation.
  4. Gel electrophoresis of plasmid and PCR product.

September 30

  1. PCR amplification of fragments 1-4 of Scl2.28 for Gibson Assembly.
  2. PCR amplification of ProS and LysA gene from the two auxotroph strains.
  3. Inoculated.

October

October 1

  1. Miniprep of liquid culture was performed using standard procedure.
  2. Nanodrop was used to quantify the purity of plasmid DNA.

October 5

  1. Fragment 4 was amplified for Gibson Assembly using higher annealing temperature.

October 6

  1. Gibson assembly of PCR amplified fragments.

October 7

  1. Transformation of BL21 with pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.

October 8

  1. Preparation of LB Agar and LB Broth containing Kanamycin.
  2. Single colonies of transformed BL21 were inoculated in LB Broth containing Kanamycin.
  3. Preparation of competent E. coli auxotroph strain JW2806 and JW0233 using calcium chloride solution.
  4. Transformation of auxotroph strain using pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.

October 9

  1. Miniprep of overnight liquid broth of BL21 transformed by pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.
  2. 4 mL of overnight starter culture of BL21 was added to 100 mL of LB Broth containing Kanamycin.
  3. IPTG was added to a final concentration of 1 mM once the OD reached 4-5.
  4. Growth was observed on positive control for JW2806 and JW0233 and Cam plates for both plasmids. Single colonies were not distinguishable in Kanamycin plates.
  5. Half of the induced cultures were pelleted at 6 hours.

October 10

  1. Remaining half of the induced cultures were pelleted after 18 hours.
  2. Preparation of 1.5% agarose gel.
  3. Preparation of LB broth.
  4. Preparation of buffer for protein purification.
  5. Double digestion of pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.
  6. PCR to verify successful ligation of pQE80L-Kan 2hr Double Digested + Scl2.28 T7 Ligated and pQE80L-Kan + Scl2.28 T4 Ligated.

October 11

  • Purification of Scl2.28 from cell pellet using Qiagen Ni-NTA Spin Kit.
  • SDS-PAGE of fraction from protein purification.

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