Notebook

July

7.12

  1. We entered our laboratory at the beginning of summer vocation, for the first time!

7.13-7.20

  1. Inoculate WB800 strain and E.coli
  2. Extract the genome DNA of B.subtilis-->CotB and CotE PCR electrophoresis gel purification
  3. Amplify Dsup and Tyr by PCR-->electrophoresis-->gel purification

7.21-7.25

  1. Take the Reverse PCR to get the linearized plasmid
  2. Plasmid gel purification to extract pHT01

7.26-7.29

  1. Inoculate and cultivate glycerol stock
  2. Extract plasmid pHT01 from the culture
  3. Enzyme digestion of plasmid pHT01 and take the reverse PCR

August

8.5

  1. Extract the genome DNA of B.subtilis--> Tyr PCR-->electrophoresis-->gel purification
  2. Preliminary experiment for transforming pHT01 into E.coli
  3. Cultivate E.coli for 12 h

8.6

  1. Obverse the plate spreaded yseterday--> one plate is fully successful;the other one only exist one bacterital colony which we consider it the hybrid bacterium
  2. Extract the genome DNA of B.subtilis--> CotE pcr,but failed-->Remodify cotE from previous PCR products
  3. Vector construction--> transform pHT01 into E.coli --> Inoculate E.coli in solid medium and cultured for 12 hours

8.7

  1. Inoculate single colonies cultured on solid medium into liquid culture medium
  2. CotE pcr again

8.8

  1. Transform pHT01 into E.coli
  2. Take a colony PCR using the colony we got on 8.6 but we failed

8.9

  1. Link four sequences by PCR
  2. Transform the linked vector into E.coli
  3. The transformation carried yesterday is successful.We choose five single colonies which were transformed and take a colony PCR. Only one Dsup band appears in the 25 bands(5 target band and 5 colonies)

8.10

  1. Pick colonies and shake them on a shaker for 5 hours
  2. Take a colony PCR using colonies
  3. Enzyme digestion of plasmid pHT01
  4. gel purification
  5. Preparation of competent cells

8.11

  1. Double Digestion of pHT01
  2. Transform the linked vector into E.coli
  3. Inoculate WB800 strain and detect optical density

8.12

  1. Selection of single colonies
  2. Take a colony PCR using colonies
  3. Extract pHT01 from the culture
  4. Transform the plasmid into WB800 by electrotransformation

8.13

  1. Transform the plasmid into WB800 by electrotransformation
  2. sequencing analysis to identify our genes

8.14

  1. Extract plasmid from DH5α
  2. Take the PCR to check the linked genes
  3. Gel purification
  4. Preparation of competent cell
  5. Gel purification

8.15

  1. Detect optical density(OD)

8.16

  1. Preparation of competent WB800
  2. Transform the plasmid into WB800 by electrotransformation

8.17

  1. Take a PCR to amplify target genes
  2. Double Digestion of pHT01
  3. Link genes and vector by T4 ligase
  4. Sequencing analysis to identify our genes

8.18

  1. Inoculate WB800 and DH5α
  2. Take a colony PCR
  3. Transform the plasmid into WB800 by electrotransformation

8.19-8.20

  1. Preparation of competent WB800
  2. Induce expression
  3. Insert DNA sequencing

8.21

  1. Transform the plasmid into WB800 by electrotransformation
  2. Extract plasmid
  3. Take a colony PCR
  4. Streak plate
  5. IPTG preparation
  6. IPTG induces gene expression
  7. Extract Dsup from WB800
  8. Western blot

8.22

  1. Test the result of the WB800N transformed by colony pcr
  2. Prepare the gel for Western blot

8.23

  1. Test the result of the WB800N transformed by colony pcr
  2. Western blot, incubate primary antibody

8.24

  1. Western blot, incubate secondary antibody
  2. Streak plate for the WB800N with first and second plasmid
  3. prepare spe antibiotic solution

8.25

  1. Use the WB800N with IPTG induced for tyrosinase activity test
  2. send our smaples for sequencing of the information ecoded
  3. amplify the WB800N with the first plasmid

8.26

  1. Western blot for secondary antibody
  2. Expose the WB800N with the first plasmid and its control group (plasmid without IPTG & without plasmid without IPTG) to UV light
  3. Colony pcr for the bactria with electrotransformation

8.27

  1. Design identification primers
  2. Colony pcr again with more colonies

8.28

  1. Digest the plasmid for identification
  2. Try to determine the best concentrtion for us iradiate
  3. Inoculate the sporulation medium

8.29

  1. Spores seperation and purification
  2. Digest plasmid and information sequence
  3. Link pDG1730
  4. Transfer them into DH5alpha

8.31

  1. Colony pcr
  2. Observe the number of colonies
  3. Streak plate
  4. Amplify the bacteria
  5. Make WB800N stock in glycerin
  6. Purify spores

9.1

  1. Streak plate
  2. Colony pcr to idntify the bacteria with the first plasmid

9.2

  1. Extract plasmid from bacteria
  2. Transform the plasmid into DH5α
  3. Incubate the first antibody

9.3

  1. Select the right colony with LB plate and Amp
  2. Electrotransfrmation
  3. Incubate the secondary antibody

  1. Purification of spores
  2. Colony pcr
  3. Link the plasmid pDG1730 with the information-stored spores
  4. Incubate primary antibody for spores

9.5

  1. Extract plasmids pDG1730 from the bacteria
  2. Identify the bacteria with pTH01
  3. Incubate second antibody for spores
  4. Immunofluorescence observation of the spore

9.6

  1. Colony pcr
  2. Spore seperation and purification
  3. Incubate the spores with primary antibody

9.7

  1. Colony pcr for our secondary plasmid
  2. IPTG induction
  3. Gel purification

9.8

  1. Purify the spores
  2. Irradiate the bacteria to a serias of UV light for viability assay
  3. Test the activity of tyrosinase with a serias of temperatures

9.9

  1. WB for the detection of Dsup proteins
  2. Inoculate the spore-inducing medium

9.10

  1. Verify the results of sequencing

9.11

  1. Immunofluorescence for the detection of melanin-binding-peptide on the spore surface

9.12

  1. Prepare the spore we shall use for the test tomorrow.
  2. Analyse the result of our bacteria-irridiation test
  3. SDS-PAGE for the detecion of Dsup.

9.13

  1. observe the result of our SDS-PAGE in the morning.

9.13-9.20

  1. School suddenly locked down because of a little outbreak of Covid19 pandemic on campus. Our experiments was pushed to be suspended as the rest of school shut down.

9.21

  1. Colony pcr
  2. Irradiate the bacteria to a serias of UV light for viability assay
  3. Try to break spores and extract DNA
  4. Incubate the spores with Primary antibody

9.22

  1. Colony pcr for our second plasmid
  2. IPTG induction
  3. Incubate the spores with secondary antibody

9.23

  1. Purify the spores
  2. Try to break spores and extract DNA
  3. Link the second plasmid
  4. Inoculate the spore-inducing medium
  5. Immunofluorescence to obvserve spores

9.24

  1. Purify the spores
  2. Colony pcr for our second plasmids
  3. Extract plasmidn

9.25

  1. Try to break spores and extract DNA
  2. Incubate the spores with Primary antibody

9.26

  1. Incubate the spores with Primary antibody
  2. Fluorescence flow cytometry
  3. Comet assay
  4. Detection of the effect of temperature on tyrosinase activity

9.27

  1. Colony PCR for second plasmid
  2. Detection of the effect of temperature on tyrosinase activity
  3. IPTG induction

9.28

  1. PCR of mutant Dsup
  2. Comet assay
  3. Inoculate the spore-inducing medium
  4. Transform the second plasmid into DH5α

9.29-9.30

  1. PCR of mutant Dsup again
  2. Purify spores
  3. Colony PCR for the second plasmid
  4. Inoculate the spore-inducing medium
  5. IPTG induction

10.1

  1. Take the PCR for Dsup with new designed primers.
  2. Comet assay again
  3. Agarose gel electrophoresis

10.2

  1. Purify spores
  2. Take the PCR for Dsup with new designed primers

10.3

  1. Inoculate spores with primary antibody
  2. Irradiate the bacteria to a serias of UV light for Comet assay
  3. Comet Assay!Last time!

10.4

  1. Inoculate spores with secondary antibody
  2. Fluorescence flow cytometry

10.5

  1. Irradiate the bacteria to a serias of UV light for Survival analysis
  2. Characterization of melanin binding is observed
  3. Plate the irradiated bacteria

10.6

  1. Break spores and extract DNA
  2. Genome sequencing of DNA
  3. Purify spores

10.7

  1. Break spores and extract DNA
  2. Extract the first plasmid that we rebuilt

10.8

  1. Point mutation PCR

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