Difference between revisions of "Team:JiangnanU China/Notebook"

Revision as of 14:24, 20 October 2019

JiangNan

iGEM Notebook

Overview

1.Construction of recombinant plasmid and recombinant bacteria with report gene

Morning:
Escherichia coli (10/50 mL) containing the pET28a plasmid was cultured, and the pET28a 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 pET28a plasmid to construct a recombinant vector. The pET28a 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 pET28a plasmids were used for enzymatic cleavage, Escherichia coli (10/50 mL) containing the pET28a plasmid was re-cultured, and plasmid extraction was performed the next day.

Morning:
The pET28a 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.

2.Safety training of phage experiments and discussion of human practice

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.

3.Phage preservation and purification of E.coli BL21 genome

Night:
Three bottles of E.coliBL21- 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.

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.

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.

Overnight PCR E. coli genome, nucleic acid gel validation.

4.Discussion of moot court with Professor Hu

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.

5.Construction of recombinant plasmid and recombinant bacteria with anti-phage gene

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.

6.Verification of anti-phage protein AbpAB

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.

Morning:
prepare the phage solution, transfer the E. coli 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.
Gene 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

7.Obtained transcriptomics data and performed data analysis

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.

8.Verification of phage-inducible promoters

1. Inoculate three 10 mL vials.
2. Common E. coli BL21, BL21 with plasmid, glcF-rfp on plasmid, and E.coli BL21 with putA-gfp recombinant plasmid. 100 μL is connected to a 10 mL Erlenmeyer flask.
3. 37 °C 180 shaker overnight culture

9.Purification of phage genome

Evening:
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.

10.Discussion of next step of experiment

Regular meetings in the group to discuss the next step. Decided to use the ARTP mutagenesis system to find new anti-phage genes.

11.Analysis of mutant strain to obtain anti-phage genes and verified them

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.

12.Constructed recombinant bacteria with antimicrobial peptide and verified it

Construction and effect verification of antimicrobial peptide P-1 (BBa_K628000), induced engineering bacteria containing antimicrobial peptides by IPTG, and observed growth.

13.Construction of respond circuit and characterized it

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.

14.Standardized all our components

Standardize all components. The person is responsible for learning to use an electron microscope and taking pictures of the phage.

@@ Line 179: / Line 179: @@
              Morning:
              <br/>
-             Escherichia coli (10/50 mL) containing the 28a plasmid was cultured, and the 28a plasmid was extracted using
+             <i>Escherichia coli</i> (10/50 mL) containing the pET28a plasmid was cultured, and the pET28a plasmid was extracted using
              a kit.
              <br/>
@@ Line 199: / Line 199: @@
              Morning:
              <br/>
-             The gfp gene was obtained by PCR and recovered by gel.
+             The <i>gfp</i> gene was obtained by PCR and recovered by gel.
              <br/>
-             The gfp gene was homologously recombined with the restriction enzyme 28a plasmid to construct a recombinant
+             The <I>gfp</i> gene was homologously recombined with the restriction enzyme pET28a plasmid to construct a recombinant
              vector.
-             The 28a plasmid was digested for later use.
+             The pET28a plasmid was digested for later use.
          </div>
      </div>
@@ Line 215: / Line 215: @@
              Evening:
              <br/>
-             Since all of the previously extracted 28a plasmids were used for enzymatic cleavage, Escherichia coli (10/50
+             Since all of the previously extracted pET28a plasmids were used for enzymatic cleavage, <i>Escherichia coli</i> (10/50
-             mL) containing the 28a plasmid was re-cultured, and plasmid extraction was performed the next day.
+             mL) containing the pET28a plasmid was re-cultured, and plasmid extraction was performed the next day.
          </div>
@@ Line 227: / Line 227: @@
              Morning:
              <br/>
-             The 28a plasmid was extracted using a kit.
+             The pET28a plasmid was extracted using a kit.
              <br/>
              The constructed recombinant vector was transferred to competent cells by transformation, and plated, and
@@ Line 265: / Line 265: @@
              Night:
              <br/>
-             Three bottles of BL21- PET28a (10/50 mL) were cultured and labeled a, b, and c.
+             Three bottles of <i>E.coli</i>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
+             The deposited BL21-28a-gfp <i>E. coli</i> was transferred to a large bottle for culture, and the next day, IPTG
              induction was performed to observe whether fluorescence was generated or not.
          </div>
@@ Line 279: / Line 279: @@
              Morning:
              <br/>
-             the phage was inserted into a bottle of E. coli, and the lysate was observed to be clarified in about 3
+             the phage was inserted into a bottle of <i>E. coli</i>, 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.
          </div>
@@ Line 287: / Line 287: @@
      <div class="content">
          <div class="fm_22">
-             The 28a plasmid and the E. coli BL21 genome were extracted from the kit.
+             The 28a plasmid and the <i>E. coli</i> BL21 genome were extracted from the kit.
              <br/>
-             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 <i>E. coli</i> genome extraction.
          </div>
      </div>
@@ Line 297: / Line 297: @@
      <div class="content">
          <div class="fm_22">
-             The kit failed to extract the E. coli BL21 genome.
+             The kit failed to extract the <i>E. coli</i> BL21 genome.
              <br/>
              The reason for reflection is that the column is used incorrectly, and the oscillating force is insufficient
@@ Line 308: / Line 308: @@
      <div class="content">
          <div class="fm_22">
-             Overnight PCR E. coli genome, nucleic acid gel validation.
+             Overnight PCR <i>E. coli</i> genome, nucleic acid gel validation.
          </div>
      </div>
@@ Line 343: / Line 343: @@
      <div class="content">
          <div class="fm_22">
-             Evening: Inoculate BL21-28a E. coli.
+             Evening: Inoculate BL21-28a <i>E. coli</i>.
          </div>
      </div>
@@ Line 353: / Line 353: @@
              Morning:
              <br/>
-             The plasmid was extracted and the resistance protein gene abpAB was amplified by PCR.
+             The plasmid was extracted and the resistance protein gene <I>abpAB</i> was amplified by PCR.
-             Afternoon: Run the glue to verify abpAB, glue recovery. Linked to the digested 28a plasmid.
+             Afternoon: Run the glue to verify <I>abpAB</i>, glue recovery. Linked to the digested 28a plasmid.
              Night:
              <br/>
@@ Line 387: / Line 387: @@
          <div class="fm_22">
              Morning:
-             <br/>prepare the phage solution, transfer the BL21 vial to the large bottle, raise the OD to 0.8, and
+             <br/>prepare the phage solution, transfer the <i>E. coli</i> 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.
              <br/>
-             AbpAB was transferred to two large bottles (added to the final concentration of 100 μL / mL of Amp
+             Gene <i>abpAB</i> 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.
@@ Line 425: / Line 425: @@
 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
+             analysis, we selected two inducible promoters, P<I>putA</i> and P<I>glcF</i>, which are capable of responding to phage
              infection in the early and late stages of phage infection.
          </div>
@@ Line 443: / Line 443: @@
 . Inoculate three 10 mL vials.
              <br/>
-. Common E. coli BL21, BL21 with plasmid, glcF-rfp on plasmid, and BL21 with putA-gfp recombinant plasmid.
+. Common <i>E. coli</i> BL21, BL21 with plasmid, <i>glcF-rfp</i> on plasmid, and <I>E.coli</I> BL21 with <i>putA-gfp</i> recombinant plasmid.
 μL is connected to a 10 mL Erlenmeyer flask.
              <br/>
@@ Line 465: / Line 465: @@
              Evening:
              <br/>
-             Connect the E. coli BL21 vial to prepare the reagents needed to extract the phage genome.
+             Connect the <i>E. coli</i> BL21 vial to prepare the reagents needed to extract the phage genome.
          </div>
      </div>
@@ Line 473: / Line 473: @@
      <div class="content">
          <div class="fm_22">
-. Preparation of phage pure culture. A small bottle of E. coli BL21 was transferred to a large bottle, and
+. Preparation of phage pure culture. A small bottle of <i>E. coli</i> 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.
              <br/>
-. Preparation of phage crude particles. The overnight cultured Escherichia coli BL21 was transferred to a
+. Preparation of phage crude particles. The overnight cultured <i>Escherichia coli</i> 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
@@ Line 548: / Line 548: @@
      <div class="content">
          <div class="fm_22">
-             The mutant strain of E. coli BL21 was obtained by ARTC (Atmospheric and room temperature plasma) mutagenesis
+             The mutant strain of <i>E. coli</i> 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
@@ Line 561: / Line 561: @@
              <br/>
              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
+             several mutant strains: <I>nuoE</i>, <I>yhjH</i>, <I>rzpD</i>, <I>gntR</i>, 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
+             with lactose operon respectively. The substrate microorganism <i>E. coli</i> 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.
+             plasmid, and the model determined that <I>gntR</i> had the least effect on the growth of <i>E. coli</i>.
              <br/>
              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.
+             to determine which resistance gene is better, the results show that <I>gntR</i> is best against phage.
          </div>
      </div>
@@ Line 598: / Line 598: @@
      <div class="content">
          <div class="fm_22">
-             We ligated the combined resistance protein genes（antP1-anpAB, antP2-gntR） and the kill switch P-1
+             We ligated the combined resistance protein genes（<i>antP1-anpAB</i>, <i>antP2-gntR</i>） 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.
              <br/>
-             In addition, we inoculated E. coli BL21 and recombinant E. coli BL21 -pET28a-PputA-abpAB-gntR-PglcF-P-1
+             In addition, we inoculated <i>E. coli</i> BL21 and recombinant <i>E. coli</i> BL21 -pET28a-P<I>putA</i>-<i>abpAB</i>-<i>gntR</i>-P<i>glcF</i>-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