06 OCT 2026 (RECONSTRUCTED)

To assess the growth quality of previous overnight (ON) cultures from Oct 05, thaw and dilute the library, and pour a set of selective plates (IP, MP, Tet and plain LB) to screen the libraries against control strains (E. coli HB101 and DH5α + pAT64).

• Overnight liquid cultures (controls) from Oct 05

• Thawed genomic/plasmid library stock

• Melted LB Agar base

• Antibiotic Stocks & Final Working Concentrations:

  • Tetracycline (Tet): 20 µg/mL

  • IP Antibiotic: 1 µg/mL

  • MP Antibiotic: 1 µg/mL

• Sterile Petri dishes, Bunsen burner, and cell spreaders

Library Dilution

• Thawed the library on ice.

• Working under aseptic conditions inside the fumehood, diluted the thawed library stock in sterile broth.

• Diluted each library either 1:10 or 1:2, depending on the library.

• Transferred the diluted library mixtures to a shaking incubator and incubated at 37°C for 1 hour so the cells could recover before plating.

Selective Plate Pouring & Labelling

• Melted the agar base, supplemented sections with respective selective antibiotics, and poured plates tailored for downstream verification. The plates were labelled as follows:

• Control Strain Series (HB101):

  • Poured 4 distinct selective plates containing: IP, MP, Tet, and Plain LB.

• Control Strain Series (DH5α + pAT64):

  • Poured 4 distinct selective plates containing: IP, MP, Tet, and Plain LB.

• Experimental Library Screen Series:

  • Poured 5 MP and 5 IP selective plates for the first 5 diluted libraries.

  • Poured an additional set of 2 MP and 2 IP plates to screen 2 further dilutions derived from 2 of those initial 5 libraries.

Plate Spreading & Drying

• Ignited a Bunsen burner to establish a sterile working field on the benchtop.

• Aseptically pipetted the respective liquid cultures and diluted library samples onto the surfaces of the labeled agar plates.

• Using a sterile spreader, distributed the liquid uniformly across the agar surface under the active flame.

• Left the plates cracked open in the fumehood to allow the surfaces to dry completely before inversion and overnight incubation on 37 C.

How the screen works?

Each clone in the library carries a different fragment of environmental DNA inside pAT64. When the library is spread on IP or MP, only clones whose DNA fragment gives carbapenem resistance should survive. The tetracycline plates show which cells carry the plasmid at all.

Why each control went on four plates?

• HB101 (no plasmid) should grow on plain LB only.

• DH5α + pAT64 (plasmid with no insert) should grow on LB and Tet, but not on IP or MP. This shows the plasmid by itself does not give carbapenem resistance.

• Plain LB confirms the cells were alive, so “no growth” on an antibiotic plate means the antibiotic stopped them, not that the cells were already dead.

Why dilute, and why recover?

• Diluting (1:10 or 1:2) spreads the cells thinly enough to give separate colonies instead of a solid lawn.

• The 1-hour recovery at 37°C lets freshly thawed cells recover before they meet the antibiotic, so genuinely resistant clones are not lost.

Next step

Read the plates and check the controls against the expected pattern (Oct 07).