Team:NJU-QIBEBT/wetlab/Notebook

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Notebook

Fluorescence Group

Part I. Plasmid Construction

2014.2.26

1. Transformation

Transform 6 plasmids into competence DH5 alpha respectively.

(9M-1,9M-2,9M-3,13D,T7-14A,T7-14C)

Remark: T7-14A,T7-14C are two plasmids containing T7 promoter

2. Fragment binding

Bind the sense strand and the antisense strand of DNA below.

(FadD promoter, FadD terminator, FabB promoter, FabB terminator, T7 promoter)

Binding System:

Binding Temperature:

Start heating at 95°C, and cool down with a temperature gradient to room temperature.

Remark: These fragments are synthesized in the professional company.

3. Single enzyme digest plasmids 16O .

Incubate in the chamber at the temperature of 37°C for three hours.

4. Ligation

Ligation System:

2014.2.27

1. Transformation

Competent cell: DH5alpha

Plasmid: FabB+GFP, FabB, FadD, T7

2014.3.1

1. Preparation of competent cell

Reagents: calcium chloride, magnesium chloride

Remark: Pre-treatment:

(1).Pick the single strain from the plate and culture it in 5mL LB culture. Put it in the shaking table with a speed of 200rpm, culturing overnight.

(2).Inoculate the overnight suspension culture to new LB culture by a ratio of 1:100, 37°C, 250rpm shake to OD=0.3.(About 4hours)

(3). Culture medium preparation: 500mL LB culture medium

2014.3.6

1. Transformation:

Transform the plasmid containing T7-14A DNA fragment into competent cell (DH5alpha).

2014.3.7

1. Extract plasmids which contain FadD DNA fragment.(FadD-3, FadD-4)

2. Measure the concentration of the plasmids:

FadD-3: 0.1μg/μL FadD-4: 0.08μg/μL

2014.3.12

1. Enzyme digestion

Single enzyme digest plasmids containing carrier 9M

Incubate in the chamber at the temperature of 37°C for three hours.

2. Fragment binding

Bind the sense strand and the antisense strand of DNA. (FadD)

Binding System:

3. Ligation

Carrier: 9M (Single enzyme digested by Xba I)

Inserted fragment: FadD

Ligation System: (10μL)

2014.3.13

1. Transformation

Transform the plasmid containing FadD to competent cell(DH5alpha).

2014.3.14

1. Pick single strain from the plate, put the bacteria in LB culture and shake it in the shaking table.

Remark:

Number the single strain 9M-1, 9M-2, 9M-3. They carry a fragment of FadD.

2014.3.18

1. Extract plasmids of the three strains.

2. Measure the concentration.

9M-1: 0.13μg/μL 9M-2: 0.14μg/μL 9M-3: 0.17μg/μL

3. Double enzyme digestion of the plasmids.(9M-1, 9M-2, 9M-3)

Digestion System(Volume: 20μL)

2014.3.20

1.Transformation

Transform plasmids containing RFP, double terminators respectively into competent cell(DH5alpha).

2014.3.21

1. Pick single strain from the plate transformed on 3.20, and shake in the shaking table overnight.

2014.3.22

1. Extract the plasmid of RFP, double terminators.

2014.3.24

1. Single enzyme (Xba I) digestion of plasmid containing RFP.

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

2. Ligation

Carrier: plasmid containing RFP (Single enzyme digested by Xba I)

Inserted fragment: FadD

Ligation System: (10μL)

3. Transformation of [FadD+RFP]

2014.3.25

1. Pick single strain of [FadD+RFP] from the plate and culture it overnight in the shaking table.

2. Sequencing of [FabB+GFP]

The sequence of [FabB+GFP-2] ,[FabB+GFP-5] and [FabB+GFP-6] are correct.

3. Enlarge cultivation of [FabB+GFP-2] ,[FabB+GFP-5] and [FabB+GFP-6].

2014.3.26

1. Extract the plasmid. ([FabB+GFP-2] , [FabB+GFP-5] and [FabB+GFP-6])

2. Measure the concentration.

[FabB+GFP-2]: 0.28μg/μL [FabB+GFP-5] : 0.23μg/μL [FabB+GFP-6]:0.35μg/μL

2014.3.27

1. Single enzyme digestion of [FabB+GFP-2] , [FabB+GFP-5] and [FabB+GFP-6]

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

2. Double enzyme digestion of the plasmid containing double terminators.

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

3. Ligation

Carrier: [FabB+GFP]-2,5,6 (Single enzyme digested by Spe I)

Inserted fragment: Double terminators (DT)

Ligation System: (10μL)

4. Transformation of [FabB+GFP+DT].

2014.3.28

1. Preliminary test whether the fragment of FadD is linked right in front of RFP in the 9M plasmid using PCR.

PCR System

2. Examine the PCR products by agarose gel electrophoresis.

Result: The bands below seem to be correct through the preliminary examination.

[FadD+RFP] 1-1,1-2,1-3,1-4,3-3

3. Pick the single strain of [FabB+GFP+DT] and incubate in the shaking table overnight.

2014.3.31

1. Sequencing [FabB+GFP+DT] and [FadD+RFP]

Results: [FabB+GFP+DT] 2-2-1 is completely correct.

[FadD+RFP] 1-4, 3-3 are correct.

2014.4.6

1. Incubate the bacterium in the shaking table overnight.([FadD+RFP] 1-4, [DT])

2014.4.7

1. Extract plasmids of [FadD+RFP] 1-4 and [DT].

2. Measure the concentration

The concentration is appropriate for the following experiments.

2014.4.10

1. Single enzyme (Spe I) digestion of [FadD+RFP]

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

2. Double enzyme digestion of [DT].

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

3. Ligation

Carrier: [FadD+RFP] (Single enzyme digested by Spe I)

Inserted fragment: [DT] (Double enzyme digested by Xba I and Spe I)

Ligation System: (10μL)

4. Transformation of [FadD+RFP+DT].

2014.4.11

1. Pick single strain of [FadD+RFP+DT] from the plate and culture it overnight in the shaking table.

2014.4.12

1. PCR directly using the colony to see if [DT] is linked behind RFP.

PCR System: (Volume: 10μL)

2. Agarose gel electrophoresis of the PCR products.

Result: The length of band fit with our prospects.

3. Sequencing the plasmid [FadD+RFP+DT]

Result: The sequence shows that the plasmid has contained double terminators.

2014.4.16

1. Transformation

Transform [FabB+GFP+DT], [FadD+RFP+DT] in the competent cell (BL-21).

2014.4.17

1. Pick single strain of [FadD+RFP+DT] and [FabB+GFP+DT] from the plate and incubate overnight in the shaking table.

2014.4.18

1. Conserve the two bacteria strains which contain the plasmid with the fragment of [FadD+RFP+DT] and [FabB+GFP+DT] respectively in the refrigerator with a temperature of -80°C.

2. After conservation, extract the plasmids. ([FadD+RFP+DT] and [FabB+GFP+DT])

3. Measure their concentration.

Result: The concentration is appropriate for the following experiments.

4. Double Enzyme Digestion

(1). Double enzyme digestion of [FadD+RFP+DT]

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

(2). Double enzyme digestion of [FabB+GFP+DT]

Digestion System: (Volume: 20μL)

Incubate in the chamber at the temperature of 37°C for three hours.

3. Ligation

Carrier: [FadD+RFP+DT] (Double enzyme digested by Spe I and Pst I)

Inserted fragment: [FabB+GFP+DT] (Double enzyme digested by Xba I and Pst I)

Ligation System: (10μL)

4. Transformation of [FadD+RFP+DT+FabB+GFP+DT].

2014.4.19

1.Pick the single strain to LB culture and incubate it overnight in the shaking table.

2014.4.20

1. Conserve the overnight bacteria strain with 15% glycerol in the refrigerator with a temperature of -80°C.

2. Sequencing the complete plasmid [FadD+RFP+DT+FabB+GFP+DT]

Result: The plasmid has contained every fragments constructed.

Part II. Fluorescence Detection

2014.4.24

1. Incubate the bacteria strain [FadD+RFP+DT], [FabB+GFP+DT] and [FadD+RFP+DT+FabB+GFP+DT] overnight.

2014.4.25

1. Inoculate the overnight suspension culture to 25mL LB culture in tubes whose volume is 50mL by a ratio of 1:100. And 20 tubes of bacteria culture suspension are needed for the coming experiments with time gradients.

2. Incubate the diluted bacteria suspension in the shaking table.

3. Take out the bacteria suspension according to the timetable below (hours):

0,1,2,3,4,6,7,8,12,18,24,36,48,60,72,84,96,108,120,132,144.

And conserve it in the refrigerator with a temperature of 4°C.

4. After six days (2014.4.30 finished), 20 tubes of each bacteria strain will be collected.

2014.5.2

1. Measure the concentration of bacteria.

(1).Take out the bacteria from the refrigerator and add every 100μL of each sample ([FadD+RFP+DT], [FabB+GFP+DT], [FadD+RFP+DT+ FabB+GFP+DT] , Each strain has 20 samples for the time gradients.) to colorless and transparent 96-well plate. And each sample need three repeat wells in order to make sure the statistics are reliable.

(2). Measure OD600 of every well using multifunctional ELIASA.

(3). Draw a concentration curve of each strain. (The concentration is shown on the vertical axis while time is shown on the horizontal axis.)

2. Fluorescence Detection

(1). Centrifuge the bacterium (6400rpm, 10min, 1mL) taken out from the refrigerator, discard the supernatant.

(2). Add 500μL PBS (Phosphate Buffer Saline) Solution to each tube and resuspend the bacterium.

(3). Centrifuge the bacterium (6400rpm, 10min) and discard the supernatant.

(4). Repeat (2), (3).

(5). Add 1mL PBS (Phosphate Buffer Saline) Solution to each tube and resuspend the bacterium.

(6). Add 100μL of each sample to black 96-well plate and each sample repeat three wells.

(7). Use multifunctional ELIASA to excite the fluorescence of GFP and RFP in the bacterium and record its fluorescence intensity.

GFP Excitation Wavelength: 501nm

Emission Wavelength: 515nm

RFP Excitation Wavelength: 584nm

Emission Wavelength: 607nm

Remark: As for the bacterium containing the plasmid of [FadD+RFP+DT], only red fluorescence is needed to be detected while only green fluorescence is needed to be detected in the bacterium with the plasmid of [FabB+GFP+DT]. However, both red and green fluorescence are needed to be detected in the bacterium with the complete target plasmid of [FadD+RFP+DT+ FabB+GFP+DT].

(8). Draw a fluorescence intensity of each strain. (The fluorescence intensity is shown on the vertical axis while time is shown on the horizontal axis.)

2014.5.8

1. Use the fluorescence microscope to examine whether GFP and RFP are expressed.

(1). Centrifuge the bacterium (6400rpm, 10min, 1mL) taken out from the refrigerator, discard the supernatant.

(2). Add 500μL PBS (Phosphate Buffer Saline) Solution to each tube and resuspend the bacterium.

(3). Centrifuge the bacterium (6400rpm, 10min) and discard the supernatant.

(4). Repeat (2), (3).

(5). Add 1mL PBS (Phosphate Buffer Saline) Solution to each tube and resuspend the bacterium.

(6). Add 100μL of each sample to black 96-well plate and each sample repeat three wells.

(7). Use the fluorescence microscope to excite the red fluorescence and green fluorescence according the standard operation in the instructions of the fluorescence microscope.

(8). Capture the fluorescence views under the microscope with the matched software and watch whether there is a trend of fluorescence intensity as the incubation time goes by.

2014.7.15

1. Incubate the bacteria strain [FadD+RFP+DT], [FabB+GFP+DT] and [FadD+RFP+DT+FabB+GFP+DT] overnight.

2014.7.17

1. Inoculate the overnight suspension culture to 25mL LB culture in tubes whose volume is 50mL by a ratio of 1:100. And 20 tubes of bacteria culture suspension are needed for the coming experiments with time gradients.

2. Incubate the diluted bacteria suspension in the shaking table.

3. Take out the bacteria suspension according to the timetable below (hours):

0,1,2,3,4,6,7,8,12,18,24,36,48,60,72,84,96,108,120,132,144.

And conserve it in the refrigerator with a temperature of 4°C.

4. After six days (2014.7.22 finished), 20 tubes of each bacteria strain will be collected.

2014.7.27

1. Extract RNA from the 60 samples of three bacteria strain.

2. Measure the RNA concentration of 60 samples.

2014.7.28

1. Reverse Transcription

Primer: RFP primer antisense

Primer: GFP primer antisense

RT System: (Volume: 10μL)

Temperature System

2014.7.30

1. qPCR

qPCR System: (Volume: 20μL)

Temperature System:









Trp group

2014.5.2

Revive and cultivate iced E.coli containing Trp operon

2014.5.4

Extract plasmid

2014.5.11

1.Extract RFP plasmid

2.digest the RFP and Trp operon

Using endonuclease XbaI and SpeI

3. test with agarose gel electrophoresis

2014.5.15

1.digest the RFP

Using endonuclease XbaI and SpeI

digest Trp operon

Using endonuclease SpeI

2. agarose gel electrophoresis

3. gel extraction

4. detect concentration

2014.5.16

cultivate E.coli containing Trp operon and E.coli containing the DNA fragment of RFP

2014.5.17

Extract plasmid

2014.5.24

1.digest Trp operon

Using endonuclease SpeI

digest the GFP

Using endonuclease XbaI and SpeI

2. agarose gel electrophoresis

3. gel extraction

4. connect the digested Trp operon with digested GFP

2014.5.25

Transform the connection product of digested Trp operon and digested GFP in LB culture medium with chloramphenicol

2014.5.29

cultivate E.coli containing Trp operon,E.coli containing the DNA fragment of GFP

2014.5.31

1.Extract plasmid

2.digest Trp operon

Using endonuclease SpeI

digest the GFP

Using endonuclease XbaI and SpeI

3. agarose gel electrophoresis

4. gel extraction

5. detect concentration

6. connect the digested Trp operon with digested GFP

2014.6.1

Transform the connection product of digested Trp operon and digested GFP in LB culture medium with chloramphenicol.

2014.6.2

pick out single colonies

2014.6.4

1. pick out single colonies

2. observe the plate with fluorescence microscope

2014.6.5

observe the plate with fluorescence microscope

2014.6.8

test the connection

split the E.coli and PCR

agarose gel electrophoresis

2014.6.13

1. pick out single colonies

2. observe the plate with fluorescence microscope

2014.6.25

1.Extract plasmid

2.digest Trp operon

Using endonuclease XbaI and SpeI

digest the GFP

Using endonuclease SpeI

3. agarose gel electrophoresis

4. gel extraction

5. detect concentration

6. connect the digested Trp operon with digested GFP

2014.6.28

1.digest Trp operon

Using endonuclease XbaI and SpeI

digest the GFP

Using endonuclease SpeI

2. agarose gel electrophoresis

2014.6.29

1.Extract plasmid

2. digest Trp operon

Using endonuclease SpeI

digest the GFP

Using endonuclease XbaI and SpeI

3. agarose gel electrophoresis

4. gel extraction

5. detect concentration

6. connect the digested Trp operon with digested GFP

2014.7.9

transform the connection product of digested Trp operon and digested GFP in LB culture medium with chloramphenicol.

2014.7.10

pick out single colonies

2014.7.11

1. Extract plasmid

2. detect concentration

2014.7.13

1. Extract plasmid

2. detect concentration

3. Conserve the plasmid with glycerinum in 193K refrigerator

2014.7.14

Test the connection

Digest using XbaI

agarose gel electrophoresis

2014.7.17

1.Test the connection

PCR

with contrast of Trp operon

2. agarose gel electrophoresis

3. send plasmid for sequencing

2014.7.23

1.connect the single digested Trp operon with double digested GFP

2. transform the connection product of digested Trp operon and digested GFP in LB culture medium with chloramphenicol

2014.7.25

pick out single colonies

2014.7.28

1. Extract plasmid

2. detect concentration

3. Conserve the plasmid with glycerinum in 193K refrigerator

2014.7.29

1.Test the connection

PCR

2. agarose gel electrophoresis

2014.8.1

Revive and cultivate iced E.coli containing Trp operon

2014.8.4

1.digest Trp operon

Using endonuclease XbaI and SpeI

2. digest the GFP

Using endonuclease SpeI

2014.8.5

1. agarose gel electrophoresis

2. gel extraction

3. detect concentration

4. connect the digested Trp operon with digested GFP

5. transform the connection product of digested Trp operon and digested GFP in LB culture medium with chloramphenicol.

2014.8.6

pick out single colonies

2014.8.7

1. send plasmid for sequencing

2. pick out single colonies with green fluorescence

2014.8.8

1.Conserve the plasmid with glycerinum in 193K refrigerator

2. observe the plate with fluorescence microscope

2014.8.9

1. pick out single colonies

2. digest Trp operon

Using endonuclease XbaI and SpeI

digest the GFP

Using endonuclease SpeI

3.transform the plasmid containing DNA fragment of GFP in LB culture medium with chloramphenicol

2014.8.10

1.Test the connection

PCR

2. agarose gel electrophoresis

2014.8.11

1.digest Trp operon

Using endonuclease XbaI and SpeI

digest the GFP

Using endonuclease SpeI

2. agarose gel electrophoresis

2014.8.25

1. dissolve new Trp operon and detect the concentration

2. digest the GFP

Using endonuclease XbaI and SpeI

3. agarose gel electrophoresis

4. gel extraction

5. detect concentration

6. connect the digested Trp operon with digested standard plasmid backbone

2014.8.27

transform the plasmid containing connection product in LB culture medium with chloramphenicol

2014.8.28

pick out single colonies

2014.9.3

1.anneal and combine two single-strand Trp operon 2.digest Trp operon

Using endonuclease Q.XbaI

Digest 16O plasmid

Using endonuclease Q.XbaI

3. agarose gel electrophoresis

4. gel extraction

2014.9.7

extract FabA and FabB,connect them with the standard plasmid

1. split the E.coli and extract the FabA and FabB fragment with PCR.

2. digest FabA and FabB with enzymes Q.XbaI and Q.SpeI

3. agarose gel electrophoresis

4. gel extraction

5. connect the digested FabA and FabB with standard plasmid backbone.

6. transform the connection product in LB culture medium with

chloramphenicol

transform the connection product of Trp operon and 16O in LB culture medium with chloramphenicol

2014.9.8

1. Observe the growing situation of E.coli

2. pick out single colonies from the E.coli transformed with the connection product of Trp operon and 16O

2014.9.9

1.PCR test the E.coli transformed with Trp operon and 16O .

2.select single colony from the E.coli transformed with connection product of FabA and FabB and standard plasmid backbone

3. prepare for sequencing

2014.9.10

1. conservate single colony from the E.coli transformed with connection product of FabA and FabB and standard plasmid backbone.

2. PCR test the E.coli transformed with connection product of FabA and FabB and standard plasmid backbone.

3. prepare for sequencing

(1).extract the plasmid MsbA-FadD XPM (2). digest the plasmid of 1 Using endonuclease EcoRI and SpeI (3). anneal and digest two single-strand Trp operon Using endonuclease Q.XbaI and Q.SpeI

4. split the E.coli and extract the FabA and FabB fragment with PCR.

2014.9.15

1. anneal and combine two single-strand Trp operon 2.digest Trp operon

Using endonuclease XbaI and SpeI

2. digest the GFP

Using endonuclease SpeI

3. agarose gel electrophoresis

4. gel extraction

5. detect concentration

6. connect the digested Trp operon with digested GFP

2014.9.15

1. digest FabA and FabB

Using endonuclease Q.XbaI and Q.SpeI

2. agarose gel electrophoresis

3. gel extraction

4. connection

(1) the digested FabA and FabB with standard plasmid backbone.

(2) the digested Trp operon with GFP.

5. transform the connection product

(1) With the contrast of GFP digested by XbaI and SpeI in LB culture medium with chloramphenicol

(2) With the contrast of GFP digested by XbaI in LB culture medium with chloramphenicol

3. Operon group

2014.6.30

1. Enzyme digestion on the lac operon

The restriction digestion system is consisted of 10 μl reagents.

Use quick cutting enzyme,on the restriction site Spe I and Pst I.

2. Enzyme digestion on the Carrier of 16O

Use quick cutting enzyme,on the restriction site Xba I and Pst I.

3.Recovery of enzyme digestion products

2014.7.2

Use the ligase solution I to link the recovery products of lac and 16O .

The restriction digestion system is consisted of 10 μl reagents.

2014.7.4

Transformation of competent cells

Use the ligated products(July 2nd) to transform the competent cells.

2014.7.5

Pick out single colonies from the plate transformed in July 4th to culture in the LB medium.

2014.7.6

Induce the lac operon using Lactose.

2014.7.7

Test the expressed products by Q-pcr.

2014.8.2

1.Enzyme digestion on the operon of ara

The restriction digestion system is consisted of 10 μl reagents.

Use quick cutting enzyme,on the restriction site Spe I and Pst I.

2.Enzyme digestion on the Carrier of 16O

Use quick cutting enzyme,on the restriction site Xba I and Pst I.

3. Recovery of enzyme digestion products

2014.8.4

Use the ligase solution I to link the recovery products of ara and 16O .

The restriction digestion system is consisted of 10 μl reagents.

2014.8.6

Transformation of competent cells

Use the ligated products(August 4th) to transform the competent cells

2014.8.8

Pick out single colonies from the plate transformed in August 6th to culture in the LB medium.

2014.8.9

Induce the ara operon using arabinose.

2014.8.10

Test the expressed products by Q-pcr.

4. experiment in QIBEBT

2014.9.17

1. Make 5 bottles of LB mediun ,9 bottles of M9 medium(500ml/bottle)

2. Activate bacterium

2014.9.18

1.Inoculate Lac-RBS-AtFatA-Term, BL21 wild-type, ara-RBS-BTE-Term seperately in LB mediun and M9 medium(doing each kind of inculation 6 bottles) with the proportion 1:100.

For every kind of bacterium ,we induced 3 of them, did not induce the rest.

2.begin to culture at 13:50,induce at 20:00 overnight,add 50μL IPTG to Lac-RBS-AtFatA-Term medium,add 100μL 1mol/L Ara to Lac-RBS-AtFatA-Term medium.

2014.9.19

Extract total lipid of the all the bacterium transformed Lac-RBS-AtFatA-Term 、ara-RBS-BTE-Term..and BL21 wild-type bacterium.

2014.9.20

1.Methyl esterificate the total lipid extracted.

2.GC-MS test.

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