1. Disposition of LB Culture Base

Goal: To create a functional and nutritional liquid environment for bacteria to grow. Liquid media used for various purposes such as propagation of microogranims or othe tests.

Materials:

1） Sterlized duran bottle, cap

2） Autoclave

3） Tryptone

4） Yeast Extract

5） Sodium Chloride

6） Double distilled H2O

7） Lab Refrigerator

Procedures:

1） Create LB Medium (Used for maintaining and cultivating recombinant straints of E.coli):

2） Prepare 14g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride= 2:1:1)

3） Add into sterlized 1L duran bottle

4） Add double distilled H2O to total volume of 700ml

5） Close the cap and autoclave it for 20 minutes at least

6） Wait until it cools

7） Store at 4 degree Celcius

2. The construction of plasmid

2.1 Construction of pTRIP

A. Obtaining target gene fragments by PCR

Goal: To amplify pTracK99 and RIP fragments for later recombination

Materials:

1） 2xPrimeMixStar*

2） pTracK99 and RIP gene template

3） double distilled water (ddH2O）

4） Primer-R

5） Primer-F

6） centrifuge tube

7) PCR thermal cycler

* PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min; the time varies with different templates

Procedure:

1） Add 2xPrimerMixStar 25µl, pTracK99 template 1µl, primer-R 1µl, and primer-F 1µl to centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2） Insert mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3） Perform 30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension.

4） Extend the fragments for 5 minutes at 72˚C

5） If not use immediately, preserve the fragments at 4˚C

6） Repeat step 1-5 for RIP. Replace pTracK99 template and the corresponding primers for those of RIP. Annealing temperature for pTracK99 is 48˚C

B. Agarose gel construction, deployment and recycle

Goal: To produce a functional and well-shaped gel for electrophoresis

Materials:

1） Casting Tray

2） Well comb

3） Microwave

4） 1x TAE

5） Agarose M

6） 10000x Nucleic acid gel stain

7） Erlenmeyer flask

Procedure:

1. Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2. Add TAE (Running buffer)

3. Heat the material with microwave

4. Melt the mixture at 30 sec interval

5. See whether bubbles are forming, if did succesfully mixed

6. Add 3μl nucleic acid gel stain (10000x)

7. Let it cool for 5~10 minutes

8. Pour the agarose mixture into the casting tray

9. Place the appropriate well comb to create the wells

10. Wait to solidify 7~10 minutes

11. Remove the comb and place the gel in the gel box

C. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to extract the bands that are of the correct length (corresponding with the length of pTRIP), eliminating the genetic fragments that didn't successfully undergo PCR.

Materials:

1） Sample

2) Marker (DNA ladder)

3) Prepared gel (from previous step)

4) 10x Loading buffer (dye)

5) Horizontal gel electrophoresis system

Procedure:

1) extract 50µl of every sample; add 5µl of loading buffer (10x) to each.

2) Place the gel in the horizontal electrophoresis system

3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform

4) Add the marker to the first well and load 50µl of each sample into the wells

5) The procedure runs at 160V for 30min

6) Afterwards, take out the gel from the device and proceed to following steps

D. DNA Gel Extraction

Goal: To recover DNA fragments in agarose gel.

Materials

l agarose gel with pTRIP

l clean scalpel

l 1.5 mL Eppendorf (EP) tube

l pipettes and sterilized pipette heads

l hot water bath

l buffer B2

l purification column and collection tube

l centrifuge

l wash solution containing pure ethanol

l elution buffer

Procedure

1) Cut the slice of gel containing pTRIP, cutting off as much unneeded gel as possible, and place it in an 1.5 mL Eppendorf (EP) tube.

2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.

3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 8000 g for 30 seconds.

4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5) Repeat above.

6) Perform centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate.

7) Transfer purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.

8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9) Discard the purification column. Store DNA in the EP tube at 4°C.

E. Homologous recombination of pTrac99k with RIP

Goal: creating the vector pTRIP as template for homologous recombination with EGFP and ccdB

Materials & Apparatus:

l ClonExpress Mix (2x)

l double distilled water (ddH₂O)

l PCR products of pTrac99k & RIP

l Nanodrop

l water bath

Procedure:

1. test the concentration on pTrac99k and RIP using Nanodrop

2. prepare the system (20μl)

3. place the system in 50°C water bath for 10min

F. Transformation

Materials

Ligated samples, LB Agar plates and liquid solution

Procedure:

a）Ligated samples are added to E.Coli samples

b）Placed on ice for 20 minutes.

c）Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

d）Add 900μL LB Agar solution, incubate for 30 minutes

e）Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f）Apply end product to agar plates, incubate for 12-16hr at 37 degrees Celsius.

*Possible mistake: It’s required to spread bacteria mildly, since harsh physical shock will damage their structure.

G. Monoclonal Antibody Validation

*purpose: DH5alpha bacteria containing constructed plasmids has multiplied. Monoclonal Antibody Validation is used for ensuring that the plasmids have replicated during this process.

Procedure

a）Within sterilized fume hood workstation, remove a colony of preprepared bacteria from the agar plate and add to 1.5mL centrifuge tube (Note: choose larger colonies but also be aware that colonies near the sides of the petri dish are most likely to be contaminated or are undesired bacteria and are less ideal)

Procedure

A. Obtain a conical flask. Use measuring cylinder to measure 100ml of (1x)TAE solution and transfer it to the conical flask.

B. Place the agarose sample on the weighing paper, Use digital balance and spatula to measure 1g of agarose sample and add it to the flask.

C. Place the flask in the microwave oven and heat in high temperature for 2 to 3 minutes.

D. Use pipette to measure 5μl of Nucleic acid dye(Gel red) and add it to the flask.

E. Add the sample in the flask to the mode of electrophoresis instrument.

F. Wait for approximately 20 minutes till the gel coagulates.

2.2 Construction of pTRIP-EGFP

A. PCR of linear pTRIP vector and EGFP

Goal: To amplify pTRIP and EGFP fragments for later recombination

Materials:

1) 2xPrimeMixStar*

2) pTRIP and EGFP gene template

3) double distilled water (ddH2O)

4) Primer-R

5) Primer-F

6) centrifuge tube

7) PCR thermal cycler

* PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min; the time varies with different templates

Procedure:

1) Add 2xPrimerMixStar 25µl, EGFP template 1µl, primer-R 1µl, and primer-F 1µl to centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2) Insert mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3) Perform 30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension.

4) Extend the fragments for 5 minutes at 72˚C

5) If not use immediately, preserve the fragments at 4˚C

6) Repeat step 1-5 to amplify pTRIP. Replace EGFP template and corresponding primers to those of pTRIP's. Instead of annealing at 55˚C, pTRIP anneals at 50˚C

7)

B. Homologous Recombination of pTRIP with EGFP

Goal: add target gene into vector so that the gene could be transformed into competent cells for expression in the future

Materials & Apparatus:

l ClonExpress Mix (2x)

l double distilled water (ddH₂O)

l PCR products of pTRIP & EGFP

l Nanodrop

l water bath

Procedure:

1. test the concentration on pTRIP and EGFP using Nanodrop

2. prepare the system (20μl)

3. place the system in 50°C water bath for 10min

C. Transformation of pTRIP-EGFP into DH5α via heatshock

Goal

By transforming the plasmids into the competent cells, quick replication of the plasmid copies are enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we are able to utilize the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids.

Transformation is conducted via the heatshock method.

Materials

The materials used are similar to the ones in the former transformation process

l LB liquid culture medium (K^-) (1ml)

l LB solid culture medium with kanamycin (K⁺)

l A box of ice

l DH5α (50μl)

l pTRIP-EGFP (10μl)

Equipments

l Water bath machine

l EP tubes

l Shaking incubator

l Mini-centrifuge

l Clean bench

Procedures

1. Take out E. coli DH5α competent cells from -80 ℃ refrigerator;

2. Add 10μl of pTRIP-EGFP and 50μl of DH5α cells into an EP tube, and 10μl of pTRIP-ccdB and 50μl of DB3.1 cells into another DH5α tube. Process repeated for 2 times in total so 2 samples of each are acquired.

3. Mix the EP tubes with the mini-centrifuge for 5 seconds

4. Put the EP tubes on ice for 30 minutes

5. Heat shock the samples at 42'C for 60 seconds in the waterbath

6. Put the EP tubes in ice for 2 minutes

7. Transfer the bacteria samples from the EP tubes into K^- liquid LB medium. This step is performed in the clean bench.

8. Shake the cultures for 1 hour at 37°C, at 220rpm

9. Spread coat 50μl on the petri dishes with K⁺ LB solid culture medium, respectively. This step is also performed in the clean bench.

10. Inverted culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

D. Plating and culturing of DH5α-pTRIP-EGFP

Goal: Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that are later going to be extracted to obtain the desired plasmid. In this case, we will culture DH5α-pTRIP-EGFP.

Materials

1) Disposable latex gloves

2) Pipette

3) Pipette tubes

4) 1L Duran bottle

5) Alcohol

6) LB medium

7) Black permanent marker

8) Sterile bacteria swabs

9) Sterile bacteria agar plates

10) Sterile collection tubes

11) Vertical clean flow bench

12) Bacteriological Incubator

Procedures

1) Label all the materials used correctly (empty plates, tube)

2) Activate the UV light in vertical clean flow bench to sterlize the environment inside

3) All the materials, including the gloves used, going into vertical clean flow bench sterlized through alcohol

4) Set up a bunsen burner--> creating sterilzed area created by the updraft of flame--> place to the right side of bench--> everytime using something, place the equipments near the fire

5) Use pipette to pour 10ml of LB medium to plates, wait until it becomes solid

6) The 0.l ml of bacteria sample (DH5α-pTRIP-EGFP) pipetted onto the surface of cooled agar plate.

7) Use sterilized bacteria swabs or spreader to spread the sample evenly over the surface of agar plate

8) Use bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria

E. Bacterium picking ( monoclonal ) and shaking

Goal: To identify if the bacteria is correct and amplify the amount of the valid strain.

l Samples collected:

EGFP 1, 2, 3, 4

l Performed under a Vertical flow clean bench.

l Protocol:

1. Add 5 ml of kanamycin-included LB medium into a 15ml incubation tube.

2. Pick colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

3. Incubate, shaking, at 37℃ for 12-16 hours.

F. Plasmid Extraction

Goal: preparing the sample so that it can go through electrophorosis to see whether it's correct

Materials & Apparatus:

l bacterial fluid of DH5α-pTRIP-EGFP

l centrifuge

l various 1.5mL ep tubes and absorption columns

l Sangon plasmid extraction kit

l pipette & pipette tubes

Procedures:

1. Take all bacterial fluid into centrifuge for 2 minutes at 8000g and abandon all supernatant

2. Add 250ul Buffer P1 to the bacteria and let it resuspend

3. Add 250 ul Buffer P2 and gently turn the centrifuge tube upside down for 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4min.

4. Add 350 ul Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix.

5. Centrifuge the tube at 12,000xg for 5-10 min, and carefully move all the supernatant into the adsorption column and centrifuge at 8,000xg for 30 sec. Drain the liquid in the collection tube.

6. Add 500ul Wash Solution into the adsorption column, centrifuge at 9,000xg for 30 sec. Drain the liquid in the collection tube.

7. repeat step 6 once

8. Put empty adsorption column and the collection tube together and centrifuge them at 9,000xg for 1 min.

9. Add 50-100ul Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min. Centrifuge it at 9,000xg for 1min.

G. Verfication of Extracted Plasmids

Goal: To produce a functional and well-shaped gel for electrophoresis

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) TAE

5) Agarose

6) Nucleic acid gel stain

7) Erlenmeyer flask

Procedure:

1. Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2. Add TAE (Running buffer)

3. Heat the material with microwave

4. Melt the mixture at 30 sec interval

5. See whether bubbles are forming, if did succesfully mixed

6. Add 3μl nucleic acid gel stain (10000x)

7. Let it cool for 5~10 minutes

8. Pour the agarose mixture into the casting tray

9. Place the appropriate well comb to create the wells

10. Wait to solidify 7~10 minutes

H. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths of the plasmids extracted from the competent cells, and thus preparing for the next step of gel extraction to extract the bands that are of the correct length (corresponding with the length of pTRIP-EGFP).

Materials:

1) Sample

2) Marker (DNA ladder)

3) Prepared gel (from previous step)

4) 10x Loading buffer (dye)

5) Horizontal gel electrophoresis system

Gel electrophoresis

1) extract 50µl of every sample; add 5µl of loading buffer (10x) to each.

2) Place the gel in the horizontal electrophoresis system

3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform

4) Add the marker to the first well and load 50µl of each sample into the wells

5) The procedure runs at 160V for 30min

6) Afterwards, take out the gel from the device and proceed to following steps

I. DNA Gel Extraction

Goal: To recover DNA fragments in agarose gel.

Materials

l agarose gel with pTRIP-EGFP

l clean scalpel

l 1.5 mL Eppendorf (EP) tube

l pipettes and sterilized pipette heads

l hot water bath

l buffer B2

l purification column and collection tube

l centrifuge

l wash solution containing pure ethanol

l elution buffer

Procedure

1) Cut the slice of gel containing pTRIP-EGFP, cutting off as much unneeded gel as possible, and place it in an 1.5 mL Eppendorf (EP) tube.

2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.

3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 8000 g for 30 seconds.

4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5) Repeat above.

6) Perform centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate.

7) Transfer purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.

8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9) Discard the purification column. Store DNA in the EP tube at 4°C.

2.3. Construction of pTRIP-ccdB

A. PCR of linear pTRIP vector and ccdB

Goal: To amplify pTRIP and ccdB fragments for recombination

Materials:

1) 2xPrimeMixStar*

2) pTRIP and ccdB gene template

3) double distilled water (ddH2O)

4) Primer-R

5) Primer-F

6) centrifuge tube

7) PCR thermal cycler

* PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min; the time varies with different templates

Procedure:

1) Add 2xPrimerMixStar 25µl, ccdB template 1µl, primer-R 1µl, and primer-F 1µl to centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2) Insert mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3) Perform 30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension.

4) Extend the fragments for 5 minutes at 72˚C

5) If not use immediately, preserve the fragments at 4˚C

6) Repeat step 1-5 to amplify pTRIP. Replace ccdB template and corresponding primers to those of pTRIP's. Instead of annealing at 55˚C, pTRIP anneals at 50˚C

B. Homologous Recombination of pTRIP with ccdB

Goal: add target gene into vector so that the gene could be transformed into competent cells for expression in the future

Materials & Apparatus:

l ClonExpress Mix (2x)

l double distilled water (ddH₂O)

l PCR products of pTRIP & ccdB

l Nanodrop

l water bath

Procedure:

1. test the concentration on pTRIP and ccdB using Nanodrop

2. prepare the system (20μl)

3. place the system in 50°C water bath for 10min

C. Transformation of pTRIP-ccdB into DB3.1 via heatshock

Goal

By transforming the plasmids into the competent cells, quick replication of the plasmid copies are enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we are able to utilize the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids. Since DH5α cannot stand the toxicity the ccdB gene induces, DB3.1, an alternative competent cell that is able to withstand the toxicity, is used. Transformation is conducted via the heatshock method, again.

Materials

The materials used are similar to the ones in the former transformation process

l LB liquid culture medium (K^-) (1ml)

l LB solid culture medium with kanamycin (K⁺)

l A box of ice

l DB3.1 (50μl)

l pTRIP-EGFP (10μl)

Equipments

l Water bath machine

l EP tubes

l Shaking incubator

l Mini-centrifuge

l Clean bench

Procedures

1. Take out E. coli DB3.1 competent cells from -80 ℃ refrigerator;

2. Add 10μl of pTRIP-EGFP and 50μl of DB3.1 cells into an EP tube, and 10μl of pTRIP-ccdB and 50μl of DB3.1 cells into another DB3.1 tube. Process repeated for 2 times in total so 2 samples of each are acquired.

3. Mix the EP tubes with the mini-centrifuge for 5 seconds

4. Put the EP tubes on ice for 30 minutes

5. Heat shock the samples at 42'C for 60 seconds in the waterbath

6. Put the EP tubes in ice for 2 minutes

7. Transfer the bacteria samples from the EP tubes into K^- liquid LB medium. This step is performed in the clean bench.

8. Shake the cultures for 1 hour at 37°C, at 220rpm

9. Spread coat 50μl on the petri dishes with K⁺ LB solid culture medium, respectively. This step is also performed in the clean bench.

10. Inverted culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

D. Plating and culturing of DB3.1-pTRIP-ccdB

Goal: Goal: Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that are later going to be extracted to obtain the desired plasmid. In this case, we will culture DB3.1-pTRIP-ccdB.

Materials

1) Disposable latex gloves

2) Pipette

3) Pipette tubes

4) 1L Duran bottle

5) Alcohol

6) LB medium

7) Black permanent marker

8) Sterile bacteria swabs

9) Sterile bacteria agar plates

10) Sterile collection tubes

11) Bunsen burner

12) Vertical clean flow bench

13) Bacteriological Incubator

Procedures

1) Label all the materials used correctly (empty plates, tube)

2) Activate the UV light in vertical clean flow bench to sterlize the environment inside

3) All the materials, including the gloves used, going into vertical clean flow bench sterlized through alcohol

4) Set up a bunsen burner--> creating sterilzed area created by the updraft of flame--> place to the right side of bench--> everytime using something, place the equipments near the fire

5) Use pipette to pour 10ml of LB medium to plates, wait until it becomes solid

6) The 0.l ml of bacteria sample (DB3.1-pTRIP-ccdB) pipetted onto the surface of cooled agar plate.

7) Use sterilized bacteria swabs or spreader to spread the sample evenly over the surface of agar plate

8) Use bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria

E. Bacterium picking and shaking

Goal: To identify if the bacteria is correct and amplify the amount of the valid strain.

Samples collected:

ccdB: 1,2,3,4

Performed under a Vertical flow clean bench.

Protocol:

1. Add 5 ml of kanamycin-included LB medium into a 15ml incubation tube.

2. Pick colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

3. Incubate, shaking, at 37℃ for 12-16 hours.

F. Plasmid Extraction

Goal: preparing the sample so that it can go through electrophorosis to see whether it's correct

Materials & Apparatus:

l bacterial fluid of DB3.1-pTRIP-ccdB

l centrifuge

l various 1.5mL ep tubes and absorption columns

l Sangon plasmid extraction kit

l pipette & pipette tubes

Procedures:

1. Take all bacterial fluid into centrifuge for 2 minutes at 8000g and abandon all supernatant

2. Add 250ul Buffer P1 to the bacteria and let it resuspend

3. Add 250 ul Buffer P2 and gently turn the centrifuge tube upside down for 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4min.

4. Add 350 ul Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix.

5. Centrifuge the tube at 12,000xg for 5-10 min, and carefully move all the supernatant into the adsorption column and centrifuge at 8,000xg for 30 sec. Drain the liquid in the collection tube.

6. Add 500ul Wash Solution into the adsorption column, centrifuge at 9,000xg for 30 sec. Drain the liquid in the collection tube.

7. repeat step 6 once

8. Put empty adsorption column and the collection tube together and centrifuge them at 9,000xg for 1 min.

9. Add 50-100ul Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min. Centrifuge it at 9,000xg for 1min.

G. Verfication of Extracted Plasmids

Goal: To produce a functional and well-shaped gel for electrophoresis

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) TAE

5) Agarose

6) Nucleic acid gel stain

7) Erlenmeyer flask

Procedure:

1. Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2. Add TAE (Running buffer)

3. Heat the material with microwave

4. Melt the mixture at 30 sec interval

5. See whether bubbles are forming, if did succesfully mixed

6. Add 3μl nucleic acid gel stain (10000x)

7. Let it cool for 5~10 minutes

8. Pour the agarose mixture into the casting tray

9. Place the appropriate well comb to create the wells

10. Wait to solidify 7~10 minutes

11. Remove the comb and place the gel in the gel box

H. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths of the plasmids extracted from the competent cells, and thus preparing for the next step of gel extraction to extract the bands that are of the correct length (corresponding with the length of pTRIP-ccdB).

Materials:

1) Sample

2) Marker (DNA ladder)

3) Prepared gel (from previous step)

4) 10x Loading buffer (dye)

5) Horizontal gel electrophoresis system

Gel electrophoresis

1) extract 50µl of every sample; add 5µl of loading buffer (10x) to each.

2) Place the gel in the horizontal electrophoresis system

3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform

4) Add the marker to the first well and load 50µl of each sample into the wells

5) The procedure runs at 160V for 30min

6) Afterwards, take out the gel from the device and proceed to following steps

I. Snap the gel to identify the length of the PCR fragment

Goal: After running electrophoresis, we need to visualize the position of the DNA bands on the gel. To do so, we used a gel transilluminator to visualize the bands and to collect the image of the gel.

Materials:

l Agarose Gel containing pTRIP-ccdB samples

l Gel transilluminator

Procedure:

1. Place the Gel inside the transilluminator

2. On a computer with a software connected to the transilluminator, visualize the results

3. On the software, take the image of the gel under UV light and save the image

4. Take the gel out and dispose it with regards to safety requirements.

5.

J. DNA Gel Extraction

Goal: To recover DNA fragments in agarose gel.

Materials

l agarose gel with pTRIP-ccdB

l clean scalpel

l 1.5 mL Eppendorf (EP) tube

l pipettes and sterilized pipette heads

l hot water bath

l buffer B2

l purification column and collection tube

l centrifuge

l wash solution containing pure ethanol

l elution buffer

Procedure

1） Cut the slice of gel containing pTRIP-ccdB, cutting off as much unneeded gel as possible, and place it in an 1.5 mL Eppendorf (EP） tube.

2） Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.

3） Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 8000 g for 30 seconds.

4） Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5） Repeat above.

6） Perform centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate.

7） Transfer purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.

8） Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9) Discard the purification column. Store DNA in the EP tube at 4°C.

3. Induction of protein expression

3.1 pTRIP-EGFP

A. Amplification culture

Goal: to acquire large amounts of target proteins for SDS-PAGE

Materials & Apparatus:

l liquid LB

l pTRIP-EGFP bacterial fluid

l kana

l inducer: N-(3-oxohexanoyl)-L-homoserine lactone

l isothermic shaker

l microplate reader

Procedure:

1. Add 100μl of pTRIP-EGFP bacterial fluid and 100μl of kana into 100mL of liquid LB

2. place the sample in the isothermic shaker 0.5h for culturing

3. Add inducers to the sample with concentrations of 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, and 1mmol respectively

4. Samples for each concentration are cultured at 22°C and 37°C for 2.5h, 5h, 10h, 20h, and 30h respectively

5. Test the concentration of all samples under the microplate reader

B. Protein Extraction and SDS-PAGE

Goal: to verify the expression of desired proteins

Protein Extraction:

Materials

l bacteria culture containing desired protein

l 50 mL centrifuge tubes

l beaker containing ice

l centrifuge

l sonicator

Procedure

1) add samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.

2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.

3) sonicate for 15 minutes.

4) repeat steps 2 and 3 for all samples.

5) perform centrifugation of samples at 4000 rpm for 15 minutes.

6) remove the cell fragments that have precipitated at the bottom of the tubes.

7) the remaining liquid will contain desired proteins.

C. SDS-PAGE:

Materials：

l Sangon 12.5% SDS-PAGE Color Preparation kit

l pipette

l distilled water

l electrophoresis buffer

l protein ladder

l vertical electrophoresis system

l Coomassie blue

Procedure：

1） using a pipette, add and mix 2.2 mL of 2X separating gel solution, 2.2 mL of 2X separating gel buffer, and 44 μL of the catalyst in a plastic cup.

2） slowly inject mixture into casting stand and frame to avoid bubbles.

3） add 1 mL water to flatten out the top.

4） wait 8 minutes for the gel to set.

5） discard the water that was added previously.

6） using a pipette, add and mix 825 μL of 2X stacking gel solution, 825 μL of 2X stacking gel buffer, and 11 μL of catalyst in a plastic cup.

7） add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles.

8） wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.

9） add the protein ladder into the first well, then load samples in each successive well.

10） transfer gel into vertical electrophoresis system.

11） set the voltage to 120V and run it for 90 minutes.

12） stain the gel with Coomassie blue by submerging it for 30 minutes.

13） EGFP is a protein that is 25 kDa; compare the protein strips to the ladder to see how well the protein has expressed.

3.2 pTRIP-ccdB

A. Amplification culture

Goal: to acquire large amounts of target proteins for SDS-PAGE

Materials & Apparatus:

l liquid LB

l pTRIP-ccdB bacterial fluid

l kana

l inducer: N-(3-oxohexanoyl)-L-homoserine lactone

l isothermic shaker

l microplate reader

Procedure:

1. Add 100μl of pTRIP-ccdB bacterial fluid and 100μl of kana into 100mL of liquid LB

2. place the sample in the isothermic shaker 0.5h for culturing

3. Add inducers to the sample with concentrations of 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, and 1mmol respectively

4. Samples for each concentration are cultured at 22°C and 37°C for 2.5h, 5h, 10h, 20h, and 30h respectively

5. Test the concentration for all samples of bacterial fluid under the microplate reader

B.Protein Extraction and SDS-PAGE

Goal: to verify the expression of desired proteins

Materials

l bacteria culture containing desired protein

l 50 mL centrifuge tubes

l beaker containing ice

l centrifuge

l sonicator

Procedure

1) add samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.

2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.

3) sonicate for 15 minutes.

4) repeat steps 2 and 3 for all samples.

5) perform centrifugation of samples at 4000 rpm for 15 minutes.

6) remove the cell fragments that have precipitated at the bottom of the tubes.

7) the remaining liquid will contain desired proteins.

C.SDS-PAGE:

Materials

l Sangon 12.5% SDS-PAGE Color Preparation kit

¡ SDS-page gel casting frame, stand, and comb

¡ 2X stacking gel solution

¡ 2X stacking gel buffer

¡ 2X separating gel solution

¡ 2X separating gel buffer

¡ catalyst for gel polymerization

¡ two single-use small plastic cups

l pipette

l distilled water

l electrophoresis buffer

l protein ladder

l vertical electrophoresis system

l Coomassie blue

Procedure

1) using a pipette, add and mix 2.2 mL of 2X separating gel solution, 2.2 mL of 2X separating gel buffer, and 44 μL of the catalyst in a plastic cup.

2) slowly inject mixture into casting stand and frame to avoid bubbles.

3) add 1 mL water to flatten out the top.

4) wait 8 minutes for the gel to set.

5) discard the water that was added previously.

6) using a pipette, add and mix 825 μL of 2X stacking gel solution, 825 μL of 2X stacking gel buffer, and 11 μL of catalyst in a plastic cup.

7) add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles.

8) wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.

9) add the protein ladder into the first well, then load samples in each successive well.

10) transfer gel into vertical electrophoresis system.

11) set the voltage to 120V and run it for 90 minutes.

12) stain the gel with Coomassie blue by submerging it for 30 minutes.

13) ccdB is a protein that is 15 kDa; compare the protein strips to the ladder to see how well the protein has expressed.

4. Functional testing

4.1 pTRIP-EGFP

A. GFP determination

Procedure

1. In 100ml LB medium, 100μL pTRIP-EGFP and pRIP ( DH5α ) bacterial solution was added, and 100μL kan was added. When the concentration was 0.5-0.8, 0.6mmol inducer ( N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ) ) was added, and the temperature was 16 degrees, 18 degrees, 22 degrees, 26 degrees, 34 degrees, 37 degrees ; after 10 hours of culture.

2. 100ul protein solution was added dropwise to the microplate, and the fluorescence intensity was measured at the excitation wavelength of 488 nm and the fluorescence detection wavelength of 525 nm.

B. Microscopic observation of fluorescent reporter gene

Procedure

1.Operation steps

2. Fluorescence observation

3. Open the bright field power switch

4. Turn on the mercury lamp switch

5. Put the sample on the stage

6. Open the fluorescent light path shutter, and close the shutter when the sample needs to be protected.

7. Adjust the optical path selection knob to the observation position

8. According to the labeling of the sample, the fluorescent filter block is rotated to the corresponding position.1.Blue light ; 2. Green light ; 3.UV ;

9. the excitation light intensity is adjusted by two groups of light-reducing filters.

10. Starting from the low power lens observation, focusing, find the pre observation field of vision.

11.In turn, change to a high-power lens to observe the sample

12.When taking pictures, the optical path selection knob is adjusted to the camera position

4.2 pTRIP-ccdB

A. Transformation of pTRIP-ccdB into E.coil DH5α、BL21

Goal

In order to detect the effect of temperature control system in E.coil DH5α and BL21.

Materials

The materials used are similar to the ones in the former transformation process

l LB liquid culture medium (K^-) (1ml)

l LB solid culture medium with kanamycin (K⁺)

l A box of ice

l E.coil DH5α、BL21 (50μl)

l pTRIP-ccdB(10μl)

Equipments

l Water bath machine

l EP tubes

l Shaking incubator

l Mini-centrifuge

l Clean bench

Procedures

1.Take out E. coli DB3.1 competent cells from -80 ℃ refrigerator;

2.Add 10μl of pTRIP-ccdB and 50μl of E.coil DH5α、BL21 . Process repeated for 2 times in total so 2 samples of each are acquired.

3.Mix the EP tubes with the mini-centrifuge for 5 seconds

4.Put the EP tubes on ice for 30 minutes

5.Heat shock the samples at 42'C for 60 seconds in the waterbath

6.Put the EP tubes in ice for 2 minutes

7.Transfer the bacteria samples from the EP tubes into K^- liquid LB medium. This step is performed in the clean bench.

8.Shake the cultures for 1 hour at 37°C, at 220rpm

9.Spread coat 50μl on the petri dishes with K⁺ LB solid culture medium, respectively. This step is also performed in the clean bench.

10.Inverted culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

B.Monoclonal verification

Samples collected:

ccdB: 1, 2, 3, 4, 5, 6, 7,8

Performed under a Vertical flow clean bench.

Protocol:

4. Add 5 ml of kanamycin-included LB medium into a 15ml incubation tube.

5. Pick colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

6. Incubate, shaking, at 37℃ for 12-16 hours.

Procedure I:

Procedure II:

C. Agarose gel electrophoresis

The steps for agarose gel electrophoresis are as follows:

1. Prepare the agarose gel: Dissolve the agarose powder in a suitable buffer solution according to the recommended concentration. Most agarose gels need to be prepared in a buffer solution.

2. Load the samples: Mix the biological macromolecule mixture (e.g., DNA samples) with a loading buffer and carefully pipette it into the wells of the prepared agarose gel.

3. Electrophoresis: Submerge the agarose gel in an electrophoresis tank filled with an appropriate electrophoresis buffer, ensuring that the samples are fully immersed. Then, connect the power supply and run the electrophoresis at the desired voltage and time.

4. Visualization and recording of results: Choose the appropriate staining method, such as DNA or protein stains, based on the type of biological macromolecules being separated. Stain the gel by immersing it in a staining solution. Finally, observe and record the migration of the biological macromolecules on the gel using a transilluminator or fluorescence imaging system.

D. The growth ability of pTRIP-cdBZ in E.coil DH5α and BL21.

Materials

l pTRIP-ccdB E.coil DH5α、BL21 (1000μl)

Procedures

1.In 100 ml LB medium, add 100μL pTRIP-ccdB bacteria liquid.

2.Add 100μLkan.

3.Culture 0D equal to 0.5 hours.

4.Adding different concentrations of inducers ( N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ) ), the concentrations were 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, 1mmol ;

5.Cultured at 22 degrees and 37 degrees for 2.5h, 5h, 10h, 20h, 30h ; the concentration of bacterial solution was measured by microplate reader at OD600.