Difference between revisions of "Team:UESTC-China/Notebook"

The school competition of iGEM in University of Electronic Science and Technology of China was held. After series of individual races and team competitions, excellent people were finally chosen into iGEM team of 2019: UESTC-China. Then we started to demonstrate the feasibility of our project in school competition and searched for some useful and interesting projects.

Through many active discussions and literature review, we decide to alleviate the ubiquitous antibiotic contamination problem by degrading ciprofloxacin. We divided all the members into three groups to explore more useful information. We actively consult relevant literature and reports to understand the current serious problem of antibiotic contamination and the existing solutions to this problem.

We continually discussed the findings of the three panelists and finally determined our plan to degrade ciprofloxacin.

We deploy the work of each member and carried out preliminary preparations, such as purchasing the necessary instruments and drugs. Meanwhile, we started to design and synthesis the plasmid.

7.8

We had transformed E.coli DH5α with plasmid BBa_K523008. Grow the transformation plates overnight at 37℃.LB liquid medium and bacterial liquid containing plasmid piGEM2018-Module001 are prepared.

7.9

We pick 4 colonies from each of the transformation plates and do Colony Polymerase Chain Reaction.And use AXYGEN kit to do plasmid extraction. Then we conduct PCR experiment with primer JZ001-A and JZ001-B to amplify the targeted section (466bp). Finally, we use DNA Agarose Gel Electrophoresis to ensure the targeted section was amplified successfully.

7.10

Gel Electrophoresis of PCR-Amplified product and get the target band we expect. Inoculate the colonies in 6mL LB medium + Chloramphenicol. Grow the cells overnight at 37℃ and 220rpm.
Bacterial transformation experiment: Add 10μL plasmid liquid into 50μL DH5α E.coli; Take 200μL transformed bacterial liquid and uniformly cover the LB plate containing Amp. Then culture it at 37℃ through the night.

7.11

Conservation for the bacteria fluid. Use AXYGEN kit to do plasmid extraction, and measure the concentration.
Test whether the plates has deteriorated.

7.12

Because the concentration is low, we repeat these experiments. We pick 4 colonies again and do Colony PCR. Then Gel Electrophoresis again and get the target band. Inoculate the right colonies in 6mL LB medium + Chloramphenicol. Grow the cells overnight at 37℃ and 220rpm.
Bacterial transformation experiment: Add 5μL plasmid liquid into 50μL TreliefTM 5α E.coli; Take 20μL/100μL transformed bacterial liquid and uniformly cover the LB plate containing Amp. Then culture it at 37℃ through the night. With the help of a postgraduate, we learn how to manipulate HPLC instrument.

7.13

Conservation for the new bacteria fluid. Use AXYGEN kit to do plasmid extraction, and measure the concentration again.
LB solid plates with Amp is prepared. And we transform the bacteria again.

7.14

Use T3 DNA polymerase to conduct colony PCR. Then we run the DNA Agarose Gel Electrophoresis. Last, the bacterial liquid is shaken at 37℃, 220rpm, cultured through the night.
The work is based on the project of 2018 UESTC-China, part BBa_K2617000, we hope to add some data to it and do some improvement.
Pick 2 colonies from each of transformation plates, and then verify that the 2018-pGEL001 has been transformed into the E.coli DH5α through polymerase chain reaction and Agarose Gel electrophoresis.
Inoculate the transformed E.coli DH5α in 6 mL LB medium + Ampicillin, and then culture overnight at 37°C and 220 rpm.

7.15

Extract the plasmid from bacteria mentioned above and the plasmid is sent to sequencing. We pay a visit to Chengdu Fourth Water Purification Plant.
Extract the plasmid 2018-pGEL001 from the bacterial fluid cultured yesterday.

7.16

Constract the new plasmid 2019-piGEMA001, which only contains iGEM2018-JSR01, the gene of bifunctional enzyme Xyn10D-fae1A.

7.17

We transform plasmids BBa_I20270 and BBa_R0040 into E.coli DH5α. Grow the transformation plates overnight at 37℃.
Try to dissolve ciprofloxacin in UP water with the concentration of 0.5mg/mL.
Transform the 2019-piGEMA001 into E.coli DH5α, and culture it in solid medium.

7.18

We pick 3 colonies from each of the transformation plates and do Colony PCR.Gel Electrophoresis of PCR-Amplified product and get the target band.Inoculate the colonies in 6mL LB medium + Chloramphenicol. Grow the cells overnight at 37℃ and 220rpm.
Pick 2 colonies from each of the transformation plates, and then verify that the 2019-piGEMA001 has been transformed into the E.coli DH5α through polymerase chain reaction and Agarose Gel electrophoresis.
Inoculate the transformed E.coli DH5α in 6 mL LB medium + Ampicillin, and culture overnight at 37°C and 220 rpm.

7.19

Conservation for the bacteria fluid. Use AXYGEN kit to do plasmid extraction, and measure the concentration. And we inoculate the colonies again.
Try to dissolve 15mg ciprofloxacin in 100mL 7% phosphoric acid. In the afternoon, 10mg ciprofloxacin is added into 400μL glacial acetic acid and 85% phosphoric acid respectively, and then the former one is volumed to 250mL precisely in a volumetric flask.
Extract the plasmid 2019-piGEMA001 from cultured bacterial fluid.
Verify that the DNA connection and transformation is right by DNA sequencing.
Conserv of the transformed E.coli DH5α.

7.20

Use AXYGEN kit to do plasmid extraction, and measure the concentration.
Transformed piGEM2019A-001 to E.coli BL21(experimental group and control group).
Make ampicillin resistant plate.

7.21

The bacteria of control group grew well. However,we found that there was no colony appearing on the plates of experimental group.
Transformed piGEM2019A-001 to E.coli BL21(experimental group and control group) again.(We failed at the first time)
Cultivated E.coli BL21 control group overnight.

7.22

We use Fluor spectrophotometer to measure the fluorescence intensity and emission peak of each bacteria containing plasmids BBa_I20270, BBa_R0040 and BBa_J364000.
Used another kind of E.coli BL21 competent cell,transformed piGEM2019A-001 to E.coli BL21(only experimental group)for the third time.
Because of the failure in the transformation of E.coli BL21 experimental group,we just used successfully transformed E.coli DH5α and E.coli BL21 to try a dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis(SDS-PAGE).
Run HPLC in our own way and get a spectrum.

7.23

E.coli BL21 experimental group grew well,so we did colony PCR and Agarose gel electrophoresis(AGE) to check if the transformation experiment succeed.
Cultivated E.coli BL21 experimental group bacteria overnight.

7.24

Used successfully transformed E.coli BL21(experimental group) and E.coli BL21(control group) to do a dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis(SDS-PAGE).(However, we did not control the same amount of bacteria (or total protein) as the control group at this time)
Dried the straw powder.

7.25

Continue the SDS-PAGE experiment and completed it.
The same amount of straw powder and the same volume of bacteria were divided into the same conical flask, and appropriate and equal amount of medium were added in, and then the culture was carried out at a constant temperature under appropriate conditions.
Learn how to use HPLC correctly under an experienced engineer’s guidance.
Pick 2 colonies from the transformation plates with plasmid BBa_J364000. Colony PCR.(Gel Electrophoresis of PCR-Amplified product and get the target band).

7.26

In order to certificate the function of lysin, we transformed E.coli DH5α with plasmid BBa_K2556051, which was obtained from ZJUT-China.
Conservation for the bacteria fluid. Use AXYGEN kit to do plasmid extraction, and measure the concentration.
Probe the minimal detectable concentration of ciprofloxacin hydrochloride
To confirm the plasmid 2019-piGEMA001 has expressed, we ultrasonic disruption of E.coli DH5α, E.coli BL21, E.coli BL21(control group) to SDS-PAGE.
Culture overnight (E.coli DH5α, E.coli BL21, E.coli BL21(control group)).

7.27

We screened the successfully transformed bacteria and carried out the colony PCR.
To combine the INPNC protein and the framework with GFP, we design plan A according to fusion PCR. And we do the first PCR step.
SDS-PAGE E.coli DH5α, E.coli BL21, E.coli BL21(control group).
The effect is not very good, but we made the determination that we only need to use E.coli BL21 and E.coli BL21(control group) since E.coli BL21 expressed plasmid 2019-piGEMA001 better.

7.28

Use USP standard ciprofloxacin hydrochloride to draw a standard curve.
We repeated colony PCR and transformation and Incubated the bacteria overnight.
We do the second step of fusion PCR. And Gel Electrophoresis of PCR-Amplified product, and get the target band. Extract DNA from Agarose Gel. Use AXYGEN enzymatic reaction kit to do DNA cleanup. Restriction Enzyme Digest using BamHⅠ.
Bradford quantified the protein, but R2 of the label of the standard curve was not>0.995 and the sample concentration was low, so it needed to be redone the next day.

7.29

We repeated colony PCR and transformation and Incubated the bacteria overnight. We also designed the follow-up experiments.
Mix the digested fragments, then do ligation with T4 ligation enzyme. Transform the ligation product into E.coli DH5α, Chl-plate.
OD quantitative bacteria amount.
Bradford changed and re-sampled the ultrasound work time to 30s.

7.30

Test whether the gradient could separate ciprofloxacin hydrochloride from Ciprofloxacin hydrochloride capsules, a kind of pharmaceuticals.
We screened the successfully transformed bacteria and carried out the colony PCR. Then they could been expanded and trained.

7.31

We measured the OD600 of the bacterial solution and performed the colony PCR again with the new reagent. Then several hours later, inducing agent was added for induction and culture.
Because there’s no bacteria on the plate, we design plan B. We redo the first step of fusion PCR. And Gel Electrophoresis of PCR-Amplified product, and get the target band. Extraction of DNA from Agarose Gel. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
SDS-PAGE 7.29‘s OD samples, but the figure is too slight to see because of the sample’s low concentration.
Continue doing yesterday‘s Bradford, but the Sample concentration is too low to SDS-PAGE.

8.1-8.4

Try to culture the E.coli TOP10 carrying the plasmid, piGEM2019-Module002. Meanwhile, we purchase standard ciprofloxacin hydrochloride monohydrate and some kinds of household drugs like the capsules, tablet, eye drops, suppository of ciprofloxacin hydrochloride.

8.5

Conserve the bacteria fluid. Use AXYGEN kit to do plasmid extraction, and measure the concentration.
We induced agent was added for induction culture. And then,we measured the OD600 of the bacterial solution every hour. Use AXYGEN kit to do plasmid extraction for fragment amplification.

8.6

We measure the standard curves of fluorescein and microspheres.

8.7

We sent the plasmid to the biological company for sequencing.
Transformation of the successfully sequence plasmids into DH5α and BL21 E.coli and culturing in inducible solid LB medium.

8.8

Ultrasonic break the bacteria fluids that one with BBa_R0040, one with BBa_J364000 and the other one with our fused plasmid. Measure the fluorescence intensity of supernatant and sediment. But no results. So we shake 50mL LB with our bacteria on the shaking table overnight at 37℃, 180r.
We consulted our instructor for experimental opinions and thought about biosafety about Horizontal codon transfer and repeated colony PCR and AGE.

8.9

Ultrasonic break 50mL bacteria fluids. Adjust the supernatant to the same OD600, so as the sediment. Measure the fluorescence intensity of supernatant and sediment. Also no results.
We prepared LB medium and prepared for tomorrow's experiment.

8.10

We redo the first step of fusion PCR. Gel Electrophoresis of PCR-Amplified product and get the target band. Extract DNA from Agarose Gel. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
We adjusted the OD600 of the bacterial solution to 0.02, measured the OD600 at one hour intervals, and made the growth curve of different experimental groups.

8.11

We pick 4 colonies again from the transformed plates. Colony PCR. Gel Electrophoresis of PCR-Amplified product and get the target band. Inoculate the colonies in 6mL LB medium + Chloramphenicol. Culture the cells overnight at 37℃ and 220rpm. Do the second step of fusion PCR, but we don’t get results.
We adjusted the OD600 of the bacterial solution to 0.05, and repeated yesterday's experiment.

8.12

Transformation of the successfully sequence plasmids into BL21 E.coli and culturing in inducible solid LB medium.

8.13

Verify the fused plasmid. But the result is wrong.
We have cultivated a large number of artificially designed synthetic suicide switches top10 E.coli on LB medium

8.14

We do PCR to verify the plasmid, but the result is not ideal.
Try to fix the single base pair which is mutant of RBS in piGEM2019-Module002.
Use AXYGEN kit to do plasmid extraction for fragment amplification. And then transform the plasmid into DH5α and transform the ara-lysin plasmid into BL21 E.coli.

8.15

Redo the fusion PCR. Do Restriction Enzyme Digestion to verify the plasmids. Make plates with Amp and CIP of different concentrations. Grow the transformed E.coil DH5α with piGEM2019-Module001 on the plates overnight.
The instructor suggested that we complete the Parts data supplement as soon as possible. The expression of Lysep3-D8 was induced by darkness, and OD600 was measured to verify its function.

8.16

We use the previous digested products to do the T4 ligation again and transformation. Redo the fusion PCR. Use AXYGEN enzymatic reaction kit to do DNA cleanup. And Restriction Enzyme Digestion using EcoRⅠand SpeⅠ.
Use AXYGEN kit to do plasmid extraction for fragment of terminator’s amplification. Gel Electrophoresis of PCR-Amplified product，and get the target band we expect. Extraction of DNA from Agarose Gel. Restriction Enzyme Digest. .Use AXYGEN enzymatic reaction kit to do DNA cleanup.

8.17

Use yesterday’s digested product to do T4 ligation and transformation.
Mix the four digested fragment with backbone of pUC57 , then do ligation with T4 ligation enzyme. And then transform the ligation product into E.coli DH5α, Amp-plate.
Transform piGEM2019-Module001 and piGEM2019-Module002 plasmids into one cell ( so-called co-transform ).

8.18

Pick 6 colonies from the plates of August 16th and 10 colonies from the plates of August 17th. Gel Electrophoresis of PCR-Amplified product, and get the target band. Inoculate the colonies in 6mL LB medium + Chloramphenicol. Grow the cells overnight at 37℃ and 220rpm.
We pick 2 colonies from each of the transformation plates and Gel Electrophoresis of PCR-Amplified product.
Determine whether HPLC could identify CIP resolved in LB

8.19

Use AXYGEN kit to do plasmid extraction, and measure the concentration. Restriction Enzyme Digestion used EcoRⅠand MfeⅠ.
We extracted the ligation product plasmid from a single colony yesterday and sent it to sequencing.

8.20

Gel Electrophoresis of yesterday’s digested products. But we don’t get the target bands. And inoculate the colonies in 6mL LB medium + Amp+ CIP. The concentrations of CIP are 0.15mg/L, 0.3mg/L, 1mg/L and 10mg/L. Grow the cells overnight at 37℃ and 220rpm.
We made the transformation with the correct plasmid and then incubated overnight.
Extract crude enzyme with ultrasonication.

8.21

Do the Restriction Enzyme Digestion of previous plasmids using SacⅠand Bgl II. Gel Electrophoresis of digest product, but don’t get the target band. Measure the OD600 of bacteria with piGEM2019-Module001.
We picked two single colonies for expansion culture: one tube under blue light, the other tube under dark conditions, and then measured their OD600s five hours later. Then SDS-PAGE was performed.
Begin to study whether the crude enzyme works.

8.22

Shake bacteria fluid transformed with piGEM2019-Module001 and another bacteria fluid transformed with JSR01 which don’t have qnrS1 on its backbone. Measure the OD600.

Use AXYGEN kit to do plasmid extraction for fragment of Gfp’s amplification.

8.23

Adjust two bacteria fluid to the same OD600. And add different concentrations of CIP into them. Shake the bacteria fluid. And measure the OD 600 per hour.
We performed a T4 connection between the digested vector and the amplified fragment. The ligation product was then transformed into DH5α competent cells and then coated with two plates.

8.24

Use the right fused plasmid to do the restriction enzyme digestion with Sac I and Bgl II. Gel Electrophoresis of digested product and get the target band.
We performed colony PCR verification on the bacteria cultured overnight, and the results were correct. So our corresponding bacteria were transferred to 6ml fresh LB liquid medium for constant temperature overnight culture.

8.25

It is a pity that our expansion has failed. We have to rebuild the vector.

8.26

We shake two control groups and adjust them to the same OD600. Add 0mg/L, 0.15mg/L, 0.3mg/L, 1mg/L, 10mg/L and 50mg/L of CIP respectively. Use the 96 wells plate to measure the OD600 per hour.
We conducted an experiment similar to 8.24 and expanded the training again.

8.27

We have successfully obtained the expanded culture liquid. We immediately extracted the plasmid DNA from it and then sequenced it.

8.28

Sequencing results show that our connection failed.
Clone CrpP-TagRFP into pEASY vector.

8.29

Shake two conical flasks of bacteria fluid with piGEM2019-Module001 and two conical flasks of control groups Adjust them to the same OD600. Add 0mg/L, 0.15mg/L, 0.3mg/L, 1mg/L, 10mg/L and 50mg/L of CIP respectively. Continue to shake them and measure the OD600 per hour.

8.30

Repeat yesterday’s experiments.

9.1

1.The plasmid JSR01-6His-CY was digested to test whether it met expectations and whether the JSR01-6His vector was successfully constructed. The result of enzyme digestion was not good, which proved that the construction of plasmid was problematic.
2. A second PCR experiment was performed to tag the end of the fragment with histidine.
3. The gel recovery experiment was carried out on the electrophoresis results of PCR products.
4. We performed a T4 connection between the digested vector and the amplified fragment. The ligation product was then transformed into DH5α competent cells and then coated with two plates.

9.2

1. The plasmid JSR01-6His-CY extracted yesterday was sent to be sequenced to find the reason why the PCR experiment failed.
2. Electrophoresis and gel recovery were performed on the products of the second PCR experiment yesterday.
3. We obtained a recombinant plasmid by ligating the lysep3-D8 gene fragment to a vector induced by IPTG. This recombinant plasmid was then transformed into E.coli BL21 strain, and plated overnight and cultured overnight.

9.3

Sequencing failed. It is suspected that the plasmid concentration is not enough. Since there was no preservation before, the transformation experiment needs to be conducted again.
We carried out the colony PCR using the E.coli DH5α strain obtained from the previous day, and the results showed that it was correct, so the corresponding single colonies were expanded and cultured.

9.4

The result of transformation experiment was not good, so the transformation experiment was carried out again.
We carried out a small-scale experiment using the bacterial culture cultured overnight, and we used the AxyPrep plasmid mini-raising kit. Finally, two tubes of plasmid DNA were obtained and sent to sequencing for verification.

9.5

Sequencing results show that our experiment failed.

9.6

1. Continue plasmid construction.
2. To study how the plasmid Peasy-CrpP can be transformed into BL21(DE3) bacteria and how to conduct induction expression experiment.

9.7

1. Continue plasmid construction.
2. The plasmid Peasy-CrpP was transformed into BL21(DE3) bacteria.

9.8

1. Continue plasmid construction.
2. Study how to use IPTG to induce the expression of Peasy-CrpP.

9.9

1. Continue plasmid construction.
2. The results were transformed by colony PCR and positive clones were selected.

9.10

1.Two positive clones were preserved with three tubes each.
2. Draw two plates, prepare for the preliminary test.
3.We have repeated the experiment of T4 connection.

9.11

1. Plasmid-JSR01 was constructed with Peasy kit.
2. Configure IPTG solution.3. We performed a single colony obtained by transforming the obtained T4 ligation product into a colony PCR. Then expand the single colony which is corret.

9.12

We carried out a small plasmid extraction with the expanded culture solution.

9.13

Culture the bacteria until the absorbance value is equal to 0.5 to obtain the approximate culture time.
We transformed the lysep3-D8 plasmid into E.coli BL21 (DE3) strain.

9.14

1. Plasmid-JSR01 was constructed with Peasy kit.
2. Culture the bacteria and prepare to induce expression.
3. We transformed the lysep3-D8 plasmid into E.coli DH5α strain.1

9.15

1. Plasmid-JSR01 was constructed with Peasy kit.
2. The induction expression experiment was conducted,but the experiment failed, red fluorescent protein was not expressed.
3. We performed colony PCR and then expanded the correct single colonies. Five hours later, the inducer was added to induce lysin gene expression and cultured overnight.

9.16

1. Plasmid-JSR01 was constructed with Peasy kit.
2. The plasmid Peasy-CrpP was transformed into BL21(DE3) bacteria again, the experiment failed.
3. We induced expression with different concentrations of inducer, then measured Abs600 every hour and plotted the growth curve.

9.17

DH5α bacteria were tried to be used to induce the expression.

9.18

1.Enzyme digestion experiments were performed to verify the correctness of the plasmid Peasy-CrpP.
2.The plasmid Peasy-CrpP was transformed into BL21(DE3) bacteria for the third time.
3. We broke the expanded culture solution, and then added the supernatant to the blank control bacterial solution to verify the function of the lysin protein.

9.19

1.The correctness of plasmid Peasy-CrpP was verified.
2. After successful transformation, colony PCR experiment was carried out and positive clones were selected for enrichment culture.
3. A large number of two different conservation strains were cultured as duplicates and prepared for tomorrow's experiments.

9.20

1. The plasmid Peasy-CrpP was extracted and verified by enzyme digestion to test whether the plasmid was successfully transformed into BL21(DE3) bacteria, but the results are not good.
2.The plasmid Peasy-CrpP was transformed into BL21(DE3) bacteria for the fourth time.

9.21

1.After successful transformation, colony PCR experiment was carried out and positive clones were selected for enrichment culture.
2.The plasmid Peasy-CrpP was transformed into BL21(DE3) bacteria for the fifth time.
3. We broke the bacterial solution that expanded the induced expression culture, and then added the supernatant to the blank control bacterial solution to verify the function of the lysin protein.

9.22

1. The plasmid Peasy-CrpP was extracted and verified by enzyme digestion to test whether the plasmid was successfully transformed into BL21(DE3) bacteria.
2. After successful transformation yesterday, colony PCR experiment was carried out and positive clones were selected for enrichment culture. The results were good.
3.We used an ultrafiltration tube to concentrate the lysin supernatant to further verify the function of the lysin. Repeat large-scale cultivation of E.coli.

9.23-9.30

1.IPTG induction expression experiment was carried out. The bacteria successfully induced and expressed were centrifuged, resuspended and whole cell was fragmented, and SDS-PAGE was carried out with whole cell fragmentation.
2.Protein purification was carried out on the cell fragmentation solution, and SDS-PAGE was carried out with the purified protein solution.
3. We used an ultrafiltration tube to concentrate the lysin supernatant to further verify the function of the lysin. Repeat large-scale cultivation of E.coli.
4. We transformed the lysep3-D8 plasmid into E.coli BL21(DE3) strain.
5. We performed colony PCR and then expanded the correct single colonies over night.
6. We carried out a small-scale plasmid experiment using the bacterial culture cultured overnight, and we used the AxyPrep plasmid mini-raising kit. Finally, 4 tubes of plasmid DNA were obtained and sent to sequencing for verification. Repeat large-scale cultivation of E.coli.
7. Sequencing results show that our plasmid concentration is very low so that sequencing failed. Repeated plasmid extraction experiment.

10.1-10.3

We successfully detected the expression of Crpp in E.coli BL21 (DE3) by SDS-PAGE.

10.4-10.10

We ultrasonically disrupted E.coli to obtain CrpP and successfully tested its function.

10.11- now

Parpering for the final presentation.