SCIENCE JOURNAL

This is my lab notebook from a microbiology research project (not independent). Each entry records what I did, why I did it and what I learned, so anyone (including future me) can follow the work step by step.

THE QUESTION

Which genes allow bacteria to survive carbapenems? Carbapenems, such as imipenem and meropenem, are among the last antibiotics that still work against many multidrug-resistant infections, and the WHO lists carbapenem-resistant Enterobacterales as a critical-priority threat.

WHY WASTEWATER?

Wastewater brings together bacteria from many different sources, along with traces of antibiotics. That makes it a hotspot where resistance genes collect and can spread between bacteria. Many of these bacteria cannot be grown in the lab, but their DNA can still be studied.

THE APPROACH: A FUNCTIONAL SCREEN

DNA from environmental bacteria is cut into fragments and cloned into the plasmid pAT64, which carries a tetracycline-resistance gene.
The plasmids are put into E. coli, creating a library of clones that each carry a different DNA fragment.
The library is spread on plates containing imipenem (IP) or meropenem (MP). Only clones whose fragment gives carbapenem resistance can grow.
Growing clones are purified by re-streaking, checked on tetracycline (to confirm they still carry the plasmid) and tested on other antibiotics such as ampicillin.
The most promising clones can then be sequenced to identify the resistance gene.

This approach is called functional metagenomics: genes are found by what they do, even if they come from bacteria that have never been grown in a lab.

CONTROLS

E. coli HB101 (no plasmid) should grow only on plain LB. This confirms the antibiotic plates stop susceptible bacteria.
E. coli DH5α + pAT64 (plasmid with no insert) should grow on LB and tetracycline, but not on IP or MP. This shows the plasmid alone does not give carbapenem resistance, and that the tetracycline plates select for the plasmid.
Plain LB plates confirm the cells were alive, so “no growth” on an antibiotic plate is meaningful.
Uninoculated broth tubes check that the media and technique are sterile.

ANTIBIOTICS USED

IP – imipenem (a carbapenem), 1 µg/mL
MP – meropenem (a carbapenem), 1 µg/mL
Tet – tetracycline, 20 µg/mL (selects for pAT64)
Amp – ampicillin (a penicillin; like the carbapenems, a β-lactam)

HOW TO READ THIS JOURNAL

Experiment – dated entries with Objective, Materials, Procedure, Observations and Discussion. The results of each day’s plates usually appear in the Observations of the next entry.
Protocol – step-by-step guides to each technique I use.

Entries marked (RECONSTRUCTED) were written up afterwards from my bench notes. To respect the lab’s confidentiality, sample sources and some details are kept general.