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

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 (466b). 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.

Straw is an agricultural byproduct of cereal plants. It makes up about half of the yield of cereal crops such as corn, barley, rice and wheat. Take corn as an example, according to FAO statistics, global corn production in 2016 was approximately 1060 million tons (Fig. 2) [2]. Every kilogram of harvested grain produces 1.2 kg of corn stalks, giving an estimate global corn straw production is 1272 million tons in 2016.

Straw is an agricultural byproduct of cereal plants. It makes up about half of the yield of cereal crops such as corn, barley, rice and wheat. Take corn as an example, according to FAO statistics, global corn production in 2016 was approximately 1060 million tons (Fig. 2) [2]. Every kilogram of harvested grain produces 1.2 kg of corn stalks, giving an estimate global corn straw production is 1272 million tons in 2016.

Straw is an agricultural byproduct of cereal plants. It makes up about half of the yield of cereal crops such as corn, barley, rice and wheat. Take corn as an example, according to FAO statistics, global corn production in 2016 was approximately 1060 million tons (Fig. 2) [2]. Every kilogram of harvested grain produces 1.2 kg of corn stalks, giving an estimate global corn straw production is 1272 million tons in 2016.

Straw is an agricultural byproduct of cereal plants. It makes up about half of the yield of cereal crops such as corn, barley, rice and wheat. Take corn as an example, according to FAO statistics, global corn production in 2016 was approximately 1060 million tons (Fig. 2) [2]. Every kilogram of harvested grain produces 1.2 kg of corn stalks, giving an estimate global corn straw production is 1272 million tons in 2016.