Team:JiangnanU China/Notebook
Morning:
Escherichia coli (10/50 mL) containing the 28a plasmid was cultured, and the 28a plasmid was extracted using a kit.
Afternoon:
The plasmid extracted in the morning was digested. The effect after the first digestion was not good. After reflection, it was considered that we are not familiar with the experimental operation. The ethanol was not dried during the rubber recovery process, and it was not carried out after the addition of the elution buffer. The recovery efficiency caused by maintaining at room temperature is lowered, so a second digestion is carried out to obtain a linear plasmid, and gel recovery is performed.
Escherichia coli (10/50 mL) containing the 28a plasmid was cultured, and the 28a plasmid was extracted using a kit.
Afternoon:
The plasmid extracted in the morning was digested. The effect after the first digestion was not good. After reflection, it was considered that we are not familiar with the experimental operation. The ethanol was not dried during the rubber recovery process, and it was not carried out after the addition of the elution buffer. The recovery efficiency caused by maintaining at room temperature is lowered, so a second digestion is carried out to obtain a linear plasmid, and gel recovery is performed.
Morning:
The gfp gene was obtained by PCR and recovered by gel.
The gfp gene was homologously recombined with the restriction enzyme 28a plasmid to construct a recombinant vector. The 28a plasmid was digested for later use.
The gfp gene was obtained by PCR and recovered by gel.
The gfp gene was homologously recombined with the restriction enzyme 28a plasmid to construct a recombinant vector. The 28a plasmid was digested for later use.
The experiment was temporarily stopped due to the lack of competent cells in the laboratory, and the team
members made a reflection.
Evening:
Since all of the previously extracted 28a plasmids were used for enzymatic cleavage, Escherichia coli (10/50 mL) containing the 28a plasmid was re-cultured, and plasmid extraction was performed the next day.
Evening:
Since all of the previously extracted 28a plasmids were used for enzymatic cleavage, Escherichia coli (10/50 mL) containing the 28a plasmid was re-cultured, and plasmid extraction was performed the next day.
Morning:
The 28a plasmid was extracted using a kit.
The constructed recombinant vector was transferred to competent cells by transformation, and plated, and cultured at 37℃ inversion.
Evening:
Preliminary observation found that the bacteria in the transgenic culture may be Bacillus subtilis, ready to be re-transformed.
The 28a plasmid was extracted using a kit.
The constructed recombinant vector was transferred to competent cells by transformation, and plated, and cultured at 37℃ inversion.
Evening:
Preliminary observation found that the bacteria in the transgenic culture may be Bacillus subtilis, ready to be re-transformed.
Afternoon:
The team will hold a group discussion to clarify the experimental precautions. For instance, all the laboratory apparatus must be sterilized before used in the next step. And discuss the HP content.
The team will hold a group discussion to clarify the experimental precautions. For instance, all the laboratory apparatus must be sterilized before used in the next step. And discuss the HP content.
Night:
Three bottles of BL21- PET28a (10/50 mL) were cultured and labeled a, b, and c. The deposited BL21-28a-gfp E. coli was transferred to a large bottle for culture, and the next day, IPTG induction was performed to observe whether fluorescence was generated or not.
Three bottles of BL21- PET28a (10/50 mL) were cultured and labeled a, b, and c. The deposited BL21-28a-gfp E. coli was transferred to a large bottle for culture, and the next day, IPTG induction was performed to observe whether fluorescence was generated or not.
Night:
Morning:
the phage was inserted into a bottle of E. coli, and the lysate was observed to be clarified in about 3 hours. The lysate was aspirated for centrifugation, and the glycerol tube was stored in a -70℃ refrigerator.
Morning:
the phage was inserted into a bottle of E. coli, and the lysate was observed to be clarified in about 3 hours. The lysate was aspirated for centrifugation, and the glycerol tube was stored in a -70℃ refrigerator.
The 28a plasmid and the E. coli BL21 genome were extracted from the kit.
Glue verification of genomic PCR failed, suspected to be a problem with E. coli genome extraction.
Glue verification of genomic PCR failed, suspected to be a problem with E. coli genome extraction.
Teacher Hu Jie in Law school gave us the opinion: Due to the lag of legislation, our national laws currently
contain no relevant laws and regulations on genetic modification.
Legal workers do not understand the field of science and technology and can only promote legislation after the occurrence of controversial events.
Genetic modification is not a bad thing in the future, but it is not advisable to deprive a small number of people of their rights because of the interests of most people.
Legal workers do not understand the field of science and technology and can only promote legislation after the occurrence of controversial events.
Genetic modification is not a bad thing in the future, but it is not advisable to deprive a small number of people of their rights because of the interests of most people.
The kit failed to extract the E. coli BL21 genome.
The reason for reflection is that the column is used incorrectly, and the oscillating force is insufficient during the extraction process, resulting in no separation of the supernatant and protein precipitation, which affects the subsequent extraction results.
The reason for reflection is that the column is used incorrectly, and the oscillating force is insufficient during the extraction process, resulting in no separation of the supernatant and protein precipitation, which affects the subsequent extraction results.
Overnight PCR E. coli genome, nucleic acid gel validation.
Evening: Inoculate BL21-28a E. coli.
Morning:
The plasmid was extracted and the resistance protein gene abpAB was amplified by PCR. Afternoon: Run the glue to verify abpAB, glue recovery. Linked to the digested 28a plasmid. Night:
Carry out the transfer. The plates were incubated overnight.
The plasmid was extracted and the resistance protein gene abpAB was amplified by PCR. Afternoon: Run the glue to verify abpAB, glue recovery. Linked to the digested 28a plasmid. Night:
Carry out the transfer. The plates were incubated overnight.
Afternoon:
Colony PCR was carried out, and the flask was cultured in a small bottle, then transferred to a large bottle for cultivation. After 3 hours, IPTG was added to induce.
Night: The plate was cultured, and the phage was added dropwise so that we can observe the infection effect.
Colony PCR was carried out, and the flask was cultured in a small bottle, then transferred to a large bottle for cultivation. After 3 hours, IPTG was added to induce.
Night: The plate was cultured, and the phage was added dropwise so that we can observe the infection effect.
Morning:
prepare the phage solution, transfer the BL21 vial to the large bottle, raise the OD to 0.8, and add 500 μL of the phage preservation solution to it, shake it at 37℃ until the bacterial solution is lysed by the phage.
AbpAB was transferred to two large bottles (added to the final concentration of 100 μL / mL of Amp antibiotics), one bottle was added with IPTG induction, the other bottle without antibiotics, and both of the bottles were raised to OD of 0.8, coated.
The 28a-abpAB vial was transferred to two large vials (Kan antibiotic was added at a final concentration of 50 μL/mL), one vial was added with 0.2 mM IPTG, and the other was not added, and the culture OD was 0.8. Since the T vector expression was slow, 28a-abpAB was used as a control to verify expression after T-vector ligation.
Pour the plate, two add IPTG, two do not. Four bottles of E. coli were separately plated, and after culturing for three hours, 1 μL of phage solution was added dropwise to the center of the plate, and the plate was allowed to stand at 37℃ after standing to dry.
Evening:
The results show that the protein AbpAB has certain anti-phage ability
prepare the phage solution, transfer the BL21 vial to the large bottle, raise the OD to 0.8, and add 500 μL of the phage preservation solution to it, shake it at 37℃ until the bacterial solution is lysed by the phage.
AbpAB was transferred to two large bottles (added to the final concentration of 100 μL / mL of Amp antibiotics), one bottle was added with IPTG induction, the other bottle without antibiotics, and both of the bottles were raised to OD of 0.8, coated.
The 28a-abpAB vial was transferred to two large vials (Kan antibiotic was added at a final concentration of 50 μL/mL), one vial was added with 0.2 mM IPTG, and the other was not added, and the culture OD was 0.8. Since the T vector expression was slow, 28a-abpAB was used as a control to verify expression after T-vector ligation.
Pour the plate, two add IPTG, two do not. Four bottles of E. coli were separately plated, and after culturing for three hours, 1 μL of phage solution was added dropwise to the center of the plate, and the plate was allowed to stand at 37℃ after standing to dry.
Evening:
The results show that the protein AbpAB has certain anti-phage ability
The phage was used to infect the Escherichia coli in log phase, and the frozen ice was used for freezing at
5 minutes and 20 minutes, and the sample was sent to the company for mRNA content, thereby obtaining
transcriptomics data and searching for inducible start-up in response to phage. Finally, through data
analysis, we selected two inducible promoters, PputA and PglcF, which are capable of responding to phage
infection in the early and late stages of phage infection.
1. Inoculate three 10 mL vials.
2. Common E. coli BL21, BL21 with plasmid, glcF-rfp on plasmid, and BL21 with putA-gfp recombinant plasmid. 100 μL is connected to a 10 mL Erlenmeyer flask.
3. 37 °C 180 shaker overnight culture
2. Common E. coli BL21, BL21 with plasmid, glcF-rfp on plasmid, and BL21 with putA-gfp recombinant plasmid. 100 μL is connected to a 10 mL Erlenmeyer flask.
3. 37 °C 180 shaker overnight culture
Evening:
Connect the E. coli BL21 vial to prepare the reagents needed to extract the phage genome.
Connect the E. coli BL21 vial to prepare the reagents needed to extract the phage genome.
1. Preparation of phage pure culture. A small bottle of E. coli BL21 was transferred to a large bottle, and
the OD was cultured to 0.5-0.6. The solution was inoculated with 1 mL of phage solution for 4-6 hours until
the bacterial solution was clear. The lysate was centrifuged at 10,000 rpm for 10 min, and the supernatant
was a phage pure culture solution.
2. Preparation of phage crude particles. The overnight cultured Escherichia coli BL21 was transferred to a large vial, the inoculum amount was 1%, amplified to a log phase (OD of about 0.4), 5 mL of phage pure culture solution was added, and cultured at 37℃ for 6-8 hours to obtain a phage lysate. DNase1 and RNaseA were added to the phage lysate to a final concentration of 5 μg/mL, and the mixture was allowed to stand at 37 °C for 1 h, then NaCl was added to a final concentration of 0.1 mM, and the mixture was dissolved and dissolved in an ice bath for 1 h (overnight).
3. The overnight phage lysate was centrifuged at 12000 rpm for 20 min. The supernatant was transferred to another centrifuge tube and PEG 6000 was added to a final concentration of 10% (w/v). After fully oscillating and dissolving, it was allowed to stand at 4 °C for 1 h, and centrifuged at 12,000 rpm for 20 min to discard the supernatant. The pellet was resuspended in 500 μL TM (or ddH2O) solution and repeatedly extracted three times with an equal volume of chloroform, and centrifuged at 12,000 rpm for 10 min to remove PEG 6000 in the resuspension, and finally a crude phage particle extract was obtained.
4. Manual extraction of phage genome DNase I and RNase A were added to the purified phage particles at a final concentration of 1 μg/mL and allowed to stand at 37 ° C for 1 h to degrade the DNA or RNA of the remaining host bacteria. Then EDTA (pH 8.0) was added to a final concentration of 50 mmol/L to terminate DNase I and RNase A activities.
Add SDS to a final concentration of 0.5%, add proteinase K to a final concentration of 50 μg/mL, mix, and digest the protein at 56 °C for 2 h. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix at 10,000 rpm for 10 min, collect the supernatant
Step 4 was repeated 3 times; the supernatant was collected, an equal volume of chloroform was mixed, and 10000 rpm, 10 min, and the supernatant was collected;
Add 1/10 volume of 3 mol/L NaAc and 2 volumes of ice 95% ethanol and mix at -20 °C for at least 2 hours. 10000 rpm, 10 min, precipitated DNA;
Add 70% ethanol (200 μL) to the pellet, and invert the capped tube several times and centrifuge at 12000 rpm for 5 min to recover the DNA.
Remove the supernatant, remove the alcohol droplets from the tube wall, and dry the open centrifuge tube for 10 min at room temperature, then resuspend the DNA with TE solution.
Detection by 0.8% agarose electrophoresis (mainly see if the strip is present, whether it is single). The gel electrophoresis band was very shallow, the DNA concentration after the gel recovery was very low, and the phage genome was successfully extracted.
2. Preparation of phage crude particles. The overnight cultured Escherichia coli BL21 was transferred to a large vial, the inoculum amount was 1%, amplified to a log phase (OD of about 0.4), 5 mL of phage pure culture solution was added, and cultured at 37℃ for 6-8 hours to obtain a phage lysate. DNase1 and RNaseA were added to the phage lysate to a final concentration of 5 μg/mL, and the mixture was allowed to stand at 37 °C for 1 h, then NaCl was added to a final concentration of 0.1 mM, and the mixture was dissolved and dissolved in an ice bath for 1 h (overnight).
3. The overnight phage lysate was centrifuged at 12000 rpm for 20 min. The supernatant was transferred to another centrifuge tube and PEG 6000 was added to a final concentration of 10% (w/v). After fully oscillating and dissolving, it was allowed to stand at 4 °C for 1 h, and centrifuged at 12,000 rpm for 20 min to discard the supernatant. The pellet was resuspended in 500 μL TM (or ddH2O) solution and repeatedly extracted three times with an equal volume of chloroform, and centrifuged at 12,000 rpm for 10 min to remove PEG 6000 in the resuspension, and finally a crude phage particle extract was obtained.
4. Manual extraction of phage genome DNase I and RNase A were added to the purified phage particles at a final concentration of 1 μg/mL and allowed to stand at 37 ° C for 1 h to degrade the DNA or RNA of the remaining host bacteria. Then EDTA (pH 8.0) was added to a final concentration of 50 mmol/L to terminate DNase I and RNase A activities.
Add SDS to a final concentration of 0.5%, add proteinase K to a final concentration of 50 μg/mL, mix, and digest the protein at 56 °C for 2 h. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix at 10,000 rpm for 10 min, collect the supernatant
Step 4 was repeated 3 times; the supernatant was collected, an equal volume of chloroform was mixed, and 10000 rpm, 10 min, and the supernatant was collected;
Add 1/10 volume of 3 mol/L NaAc and 2 volumes of ice 95% ethanol and mix at -20 °C for at least 2 hours. 10000 rpm, 10 min, precipitated DNA;
Add 70% ethanol (200 μL) to the pellet, and invert the capped tube several times and centrifuge at 12000 rpm for 5 min to recover the DNA.
Remove the supernatant, remove the alcohol droplets from the tube wall, and dry the open centrifuge tube for 10 min at room temperature, then resuspend the DNA with TE solution.
Detection by 0.8% agarose electrophoresis (mainly see if the strip is present, whether it is single). The gel electrophoresis band was very shallow, the DNA concentration after the gel recovery was very low, and the phage genome was successfully extracted.
Regular meetings in the group to discuss the next step. Decided to use the ARTP mutagenesis system to find
new anti-phage genes.
The mutant strain of E. coli BL21 was obtained by ARTC (Atmospheric and room temperature plasma) mutagenesis
system, and the mutant strain and the phage were mixed and cultured for 12 h, the temperature was 37 ° C,
the shaking speed was 180 rpm, and the culture solution was coated on the culture solution. The culture dish
was cultured at 37 ° C for 24 hours at a constant temperature. A single colony capable of growing a single
colony on the plaque was a mutant strain capable of resisting phage infection. All single colonies were
inoculated into 10 mL of LB medium at 37 ° C, and the rotation speed was 180. Incubate at 180 rpm for 12
hours, then apply 100 μL of the culture solution to the plate, and add 1 μL of the phage solution to the
center of the plate. If the plaque appears after 37 ° C culture overnight, the mutant strain is eliminated.
When no plaque appeared, the plate was streaked, and single colony re-culture and plate verification were
carried out. Through 10 rounds of culture and plate verification, the mutation site and anti-phage traits
were consolidated, and 8 mutant strains were finally screened.
The whole genome sequencing of eight mutant strains was carried out to obtain the mutant genes shared by several mutant strains: nuoE, yhjH, rzpD, gntR, and these four genes were ligated to the pET-28a plasmid with lactose operon respectively. The substrate microorganism E. coli BL21 was induced by IPTG with a final concentration of 0.5 mmol/L. The induction temperature was 37 °C for 12 h. During the period, samples were taken at 0 h, 4 h, 6 h, 8 h, 10 h. Its OD600, compared with the E. coli growth curve containing the empty plasmid, and the model determined that gntR had the least effect on the growth of E. coli.
After incubation for 12 h, 100 μL of the culture solution was applied to a Petri dish containing a final concentration of 0.5 mmol/L, and then 1 μL of the phage solution was added dropwise to the center of the culture dish, and cultured at 37 ° C for 12 hours at a constant temperature to observe plaques. Transparency to determine which resistance gene is better, the results show that gntR is best against phage.
The whole genome sequencing of eight mutant strains was carried out to obtain the mutant genes shared by several mutant strains: nuoE, yhjH, rzpD, gntR, and these four genes were ligated to the pET-28a plasmid with lactose operon respectively. The substrate microorganism E. coli BL21 was induced by IPTG with a final concentration of 0.5 mmol/L. The induction temperature was 37 °C for 12 h. During the period, samples were taken at 0 h, 4 h, 6 h, 8 h, 10 h. Its OD600, compared with the E. coli growth curve containing the empty plasmid, and the model determined that gntR had the least effect on the growth of E. coli.
After incubation for 12 h, 100 μL of the culture solution was applied to a Petri dish containing a final concentration of 0.5 mmol/L, and then 1 μL of the phage solution was added dropwise to the center of the culture dish, and cultured at 37 ° C for 12 hours at a constant temperature to observe plaques. Transparency to determine which resistance gene is better, the results show that gntR is best against phage.
Construction and effect verification of antimicrobial peptide P-1 (BBa_K628000), induced engineering
bacteria containing antimicrobial peptides by IPTG, and observed growth.
We ligated the combined resistance protein genes（antP1-anpAB, antP2-gntR） and the kill switch P-1
（BBa_K628000）after the latent phage-inducible promoter and the burst-inducible promoter, and performed
the phage infection assay on the LB agar plate, and got very good resistance.
In addition, we inoculated E. coli BL21 and recombinant E. coli BL21 -pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Than the fresh phage solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the recombinant grew well.
In addition, we inoculated E. coli BL21 and recombinant E. coli BL21 -pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Than the fresh phage solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the recombinant grew well.
Standardize all components. The person is responsible for learning to use an electron microscope and taking
pictures of the phage.
