Experiments

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All experiments conducted were based on classical techniques in the fields of molecular and synthetic biology, as detailed in the protocol and notebook sections.



PCR

The initial experiment involved PCR to amplify the genetic material obtained from the synthesis carried out by IDT. This amplification was necessary to enable subsequent plasmid digestion. PCR was also employed in conjunction with agarose gels to confirm amplification and identify the genomes of the bacterial strains used.



Plasmid Digestion


To proceed with the experiments, the already amplified primers and the restriction enzymes NotI and EcoRI were used to insert the SARP gene into pSET152Ec, which exhibits constitutive expression. However, several combinations with these enzymes were required to achieve the correct model with the expected molecular weight.


Transformation of DH5α


After confirming the digestion of the plasmid, we attempted to transform DH5α bacteria to augment the quantity of available plasmids. Unfortunately, we encountered several challenges during this process, including difficulties in confirming the vector's digestion, ensuring the successful transformation of the bacteria, and facing challenges in selecting antibiotic-resistant strains from the medium. Consequently, we were unable to test the hypothesis regarding SARP regulation and the modulation necessary to enhance cosmomycin production.


Metabolic Engineering


Our experiments aimed to reconfigure the metabolic pathways of the bacteria to boost cosmomycin production by increasing the availability of malonate. Initially, we planned to synthesize a specific plasmid for this purpose. However, due to its high G-C content and size, synthesizing the plasmid proved unfeasible, even after attempting to divide it into two fragments for later assembly. Furthermore, the same issue of a high G-C base composition prevented us from producing the synthetic enzyme needed to enhance malonate production.

Used Protocols:


PCR

PCR was prepared using the high fidelity Phusion polymerase from Thermo Scientific with the following protocol:

    Primer Forward 5’-TATATGCGGCCGCGCGATCTATGGGGGAACG-3’


    Primer Reverse 5’-TATATCTTAAGTCAGTAGACCCGCACCG-3’


    30.5 µL H2O; 1 µL dNTPs; 1.5 µL DMSO; 10 µL 5x GC buffer; 2.5 µL primer foward in 10 µM concentration; 2.5 µL primer reverse in 10 µM concentration; 1.5 µL DNA template and 0.5 µL enzyme.


    The reaction was incubated as:

      30s at 98°C


      10s at 98°C*


      30s at 61°C*


      60s at 72°C*


      5 min at 72°C


      hold at 4°C

    34 cycles repeating the steps indicated by *


Digestion and Ligation of the plasmid and Transformation of bacteria

Materials:

  • Plasmid DNA
  • Restriction enzymes NotI and EcoRI
  • T4 DNA Ligase
  • Incubator or Water Bath (37°C)
  • Heat Block or Thermocycler (65°C)
  • Gel Electrophoresis Equipment
  • Agarose Gel
  • DNA Size Marker
  • Gel Extraction Kit

Protocol:

1. Digestion of Plasmid DNA and DNA Fragment(s)

1.1 Set up a master mix for each digestion reaction as follows (adjust volumes as needed):

  • 1. Plasmid DNA
  • Restriction enzymes NotI and EcoRI
  • T4 DNA Ligase

1.2. Mix gently by pipetting and centrifuge briefly to collect all liquid at the bottom of the tube.


1.3. Incubate the reaction mixtures in a 37°C incubator or water bath for the recommended time (usually 1-2 hours). Ensure that the reaction tubes are securely capped to avoid evaporation.


1.4. After digestion, inactivate the enzymes by heating the reaction mixtures at 65°C for 10-15 minutes.


2. Analyze Digestion


2.1. Perform agarose gel electrophoresis to confirm the success of the digestion. Should have distinct bands corresponding to the expected sizes.


3. Purify Digested DNA Fragments

3.1. The purification of DNA was done using Thermo Scientific GeneGET Gel extraction kit


4. Transformation of bacteria by heat shock

4.1. With a pellet of DH5α, ressuspend the bacteria in a solution of calcium chloride and incube for 30 minutes;



5. Plating and Selection

5.1. Plate the transformed cells on selective agar plates containing the appropriate antibiotic(s) to select for the desired plasmid.


6. Incubation and Analysis

6.1. Incubate the plates at the appropriate temperature for the host organism until colonies appear.


6.2. Analyze with a agarose gel electrophoresis the colonies by plasmid isolation to confirm the presence of the desired insert.