Difference between revisions of "Part:BBa K5248021"

Latest revision as of 12:00, 2 October 2024

FimE Knockout

Overview

In order to develop a collective protective mechanism centered on the production of type I fimbriae in Escherichia coli Nissle 1917. Firstly we try to knock out FimE by using the lambda red recombinase system protocol. After two failed attempts at this method, we analyzed that there may be an A/T-rich region with probability a serious secondary structure near the FimE gene in Escherichia coli Nissle 1917. Then, the mutant Escherichia coli Nissle 1917, ΔfimE, was constructed through homologous recombination of a suicide plasmid (pCVD442), and validated the knockout method in Escherichia coli DH5α.

FimE knockout

FimE forms a transient covalent Tyr linkage to DNA. FimE, along with FimB, is a recombinase in Escherichia coli which catalyzes the site-specific recombination required for inversion of a 314-bp invertible DNA element, the fim switch (fimS), to control transcription of the type I fimbrial structural genes--a process known as phase-variation switching. fimS contains the promoter for expression of the fimbrial structural genes.

Strain construction Gene deletion in Cronobacter malonaticus 3267 was done using the lambda red recombinase system protocol with minor modifications. The ΔfimE mutant overexpressed type 1 fimbriae leading to the formation of microcolonies. Target cells within these microcolonies were protected from contact-dependent killing mediated by the type 6 secretion system (T6SS), leading to their survival. This defense mechanism could represent a nonspecific resistance mechanism, unlike immunity proteins, which could potentially lead to resistance against a broad range of assailants. Reference: Margot Marie Dessartine et al. Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks. mBio. 2024 Apr 10;15(4):e0255323.

Construction of Targeting Vector and Preparation of Donor Strain:

(1) Amplification and Cloning of fimE Gene Upstream and Downstream Homologous Recombination Arms:

PCR Amplification Reaction:

1. Bacterial Suspension 0.2 μl 2. fimE-5F/3F (50pmol/μl) 0.2 μl 3. fimE-5R/3R (50pmol/μl) 0.2 μl 4. dH2O 9.4 μl 5. PrimeSTAR Max Premix(2×),TaKaRa 10 μl 6. Total 20 μl

1. 95 degree 5 min 2. 95 degree 30 sec 3. 66 degree 30 sec 4. 72 degree 60 sec 25 cycles 8. 72 degree 7 min

(2) High-Fidelity PCR Reaction to Amplify Kanamycin Resistance Gene (Kn) from pKD4 Plasmid:

PCR Amplification Reaction: 1. pKD4（10ng/ul） 0.2 μl 2. fimE-KnF (50pmol/μl) 0.2 μl 3. fimE-KnR (50pmol/μl) 0.2 μl 4. dH2O 9.4 μl 5. PrimeSTAR Max Premix(2×) 10 μl 6. Total 20 μl

1. 95 degree 5 min 2. 95 degree 30 sec 3. 66 degree 30 sec 4. 72 degree 60 sec 20 cycles 8. 72 degree 7 min

(3) Fusion PCR to Connect fimE Gene Upstream and Downstream Homologous Arms with Kn Resistance Gene:

PCR Amplification Reaction: 1. fimE-5F/5R PCR product 3.2 μl 2. fimE-3F/3R PCR product 3.2 μl 3. fimE-KnF/R PCR product 3.2 μl 4. fimE-5F (50pmol/μl) 0.2 μl 5. fimE-3R (50pmol/μl) 0.2 μl 6. PrimeSTAR Max Premix(2×) 10 μl 7. Total 20 μl

1. 95 degree 5 min 2. 95 degree 30 sec 3. 66 degree 30 sec 4. 72 degree 1 min 20 cycles 8. 72 degree 7 min

Electrophoresis Results:

M: DNA Molecular Weight Marker. From top to bottom: 2000, 1500, 1000, 700, 400, 200 bp, with 1000 bp highlighted. 1: Amplification result of upstream homologous arm, 851 bp; 2: Amplification result of downstream homologous arm, 893 bp; 3: Amplification result of kanamycin resistance gene, 1526 bp; The above 3 fragments were separated and purified by excision of DNA band from agarose gel and column centrifugation after electrophoresis (DNA gel recovery kit, Sangon Biotech), and eluted in 30 μl sterile deionized water for subsequent fusion PCR; Electrophoresis Results:

The DNA molecular weight standards are arranged from top to bottom as follows: 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 base pairs (bp), with the 750 bp band being highlighted.

1: The length of the targeting sequence (upstream homologous recombination arm - Kn resistance gene coding sequence - downstream homologous recombination arm) is 3211 base pairs (bp). (4) Construction of Targeting Vector (pCVD442-ΔfimE::Kn):

Enzymatic Digestion Reaction:

The plasmid pCVD442 was digested with SmaI restriction enzyme, followed by gel excision and purification.

pCVD442（~100ng/μl） 10μl 10Tango buffer(MBI) 5μl SmaI(10U/μl,MBI) 2μl ddH2O 33μl Total 50μl

The reaction was carried out at 30 degrees Celsius for 2 hours. After the restriction enzyme digestion, both the vector and the fusion PCR products were separated by 1% agarose gel electrophoresis, followed by column centrifugation purification, and eluted in 50 μl of deionized water Ligation and Transformation:

1.pCVD442/SmaI(~50ng/μl) 2μl 2.fimE::Kn(~50ng/μl) 6μl 3.10T4 buffer 1μl 4.T4 DNA ligase(5U/μl,MBI) 1μl 5.Total 10μl Reaction at 16°C overnight.

The ligation product is precipitated with isopropanol, washed with 70% ethanol, and dissolved in 5 μl of deionized water. It is then introduced into E. coli DH5α lpir by electroporation and cultured on LB plates containing kanamycin (50 μg/ml) at 37°C until monoclonal colonies form.

Dentification of Positive Clones and Preparation of Targeting Plasmids: Colonies growing on kanamycin double-resistant plates contain the targeting fragment. After selecting one clone and verifying the correct sequence by sequencing, subsequent experiments are carried out. The clone is named pCVD442-ΔfimE::Kn. This clone is inoculated into 3 ml of LB medium containing kanamycin (50 μg/ml) and cultured overnight at 37°C. On the following day, plasmid DNA is purified using column centrifugation. (5) Construction of Donor Strains:

The targeting vector pCVD442-ΔfimE::Kn is electroporated into E. coli β2155 strain and spread on LB plates (with ampicillin 100 μg/ml, 0.5 mM DAP). After incubation at 37°C until single colonies form, this clone is used as the donor strain for conjugation experiments, named β2155/pCVD442-ΔfimE::Kn.

Conjugation Experiment and Screening of fimE Gene Knockout Strain:

(1) Inoculation of recipient strain E. coli Nissle1917 on LB plates and incubation at 37°C until single colonies form. Single colonies are then inoculated into 3 ml of LB and cultured overnight at 37°C, 220 rpm.

(2) Single colonies of β2155/pCVD442-ΔfimE::Kn are inoculated into 3 ml of LB (with ampicillin 100 μg/ml, 0.5 mM DAP) and cultured overnight at 37°C, 220 rpm.

(3) 500 μl of donor strain β2155/pCVD442-ΔfimE::Kn and 500 μl of recipient strain (Nissle1917) are mixed for conjugation.

(4) 50 μl of the conjugated culture is spread on LB plates with kanamycin (50 μg/ml) and incubated at 30°C until single colonies form.

(5) Randomly selected clones from the antibiotic plate are picked and cultured in 100 μl of LB. After mixing, 10 μl is inoculated into 3 ml of LB and cultured overnight at 30°C, 220 rpm. The next day, 50 μl of the culture is spread on LB plates with 10% sucrose (with kanamycin 50 μg/ml, without NaCl) and incubated at 30°C until single colonies form. Randomly selected single colonies are then cultured in 3 ml of LB (with kanamycin 50 μg/ml) overnight at 30°C. The next day, a small amount of culture is used for PCR identification with internal primers of the fimE gene. The results show that except for clones 4 and 8, all other clones are negative, indicating that the fimE gene should have been replaced, but further identification with external primers is needed.

(6) A small amount of culture from clone 1 is used for PCR reaction with external primers. The result shows a specific amplification product of the correct length (3492 bp for the replaced strain), confirming this clone as a positive clone where the fimE gene has been replaced by the kanamycin resistance gene, named Nissle1917/ΔfimE::Kn. Internal Primer Identification PCR Reaction:

1. Bacterial Suspension 0.5 μl 2. 10 Taq buffer 5 μl 3. dNTP(2.5mM) 4 μl 4. fimE-inF(50pmol/μl) 0.5 μl 5. fimE-inR(50pmol/μl) 0.5 μl 6. Taq DNA polymerase(5U/μl，MBI) 0.5 μl 7. dH2O 39 μl 8. Total 50 μl

1. 95 degree 5 min 2. 95 degree 30 sec 3. 62 degree 30 sec 4. 72 degree 30 sec

  	30 cycles

5. 72 degree 7 min

Electrophoresis Results:

M: DNA Molecular Weight Marker. From top to bottom: 1200, 900, 700, 500, 300, 100 bp, with 700 bp highlighted. 1-14: Amplification results of clones 1-14 with internal primers; 15: Amplification result of the original strain with internal primers, product length: 268 bp; 16: Negative control amplification result;

External Primer Identification PCR Reaction:

1. Bacterial Suspension 0.5 μl 2. 10 Taq buffer 5 μl 3. dNTP(2.5mM) 4 μl 4. fimE-outF(50pmol/μl) 0.5 μl 5. fimE-outR(50pmol/μl) 0.5 μl 6. Taq DNA polymerase(5U/μl，MBI) 0.5 μl 7. dH2O 39 μl 8. Total 50 μl

1. 95 degree 5 min 2. 95 degree 30 sec 3. 66 degree 30 sec 4. 72 degree 3 min

  	        35 cycles

5. 72 degree 7 min

Electrophoresis Results:

M: DNA Molecular Weight Marker From top to bottom, the molecular weights are as follows: 10,000, 8,000, 6,000, 5,000, 4,000, 3,000 (highlighted), 2,500, 2,000, 1,500, 1,000 (highlighted), 750, 500, 250 base pairs (bp).

1: Amplification result of the outer primer for clone 1, 3,492 base pairs (bp).

2: No-template negative control.

Strain Preservation: Take 600 μl of fresh culture of Nissle1917/ΔfimE::Kn, mix with an equal volume of 50% sterile glycerol to preserve the strain, and store at -80°C for long-term preservation.

Sequence and Features