• Plate 100 μl of this media on LB plates prepared before containing antibiotic, ampicillin.
These LB plates are prepared beforehand.
2.3.1. Primary culture
• 5 μl of ampicillin is added to 5 μl of LB broth in a tube.
• The LB plates are scrutinised and a single colony is identified.
• We use a teaser to add the bacteria from the plate to the tube containing ampicillin.
• In this step we make use of a control by taking another similar broth tube with ampicillin
but no bacteria.
• These two tubes are incubated overnight.
• Next morning it is observed that one of the tubes turn turbid, i.e., the one containing
bacteria, while the control does not.
• Again 100 μg/ml ampicillin is added to 100 ml broth without touching the media.
• This culture is called the primary culture.
2.3.2. Secondary culture
• 1% of primary culture bactria is aliquoted. This is the secondary culture.
• 1% of 100 ml is 1ml and hence 1 ml is aliquoted.
3. Induction of protein synthesis, gene expression in bacteria with IPTG
Control of the pTaq expression system is accomplished through the lac promoter and operator.
Before target gene can be transcribed, T7 polymerase must be present. The gene on the host cell
chromosome usually has an inducible promoter which is activated by addition of Isopropyl-beta-
thio galactopyranoside (IPTG). This molecule, IPTG, displaces the repressor from the lac operator.
Since there are lac operators on both the gene encoding T7 polymerase and target gene, IPTG
activates both genes. Therefore, when IPTG is added to the cell, the T7 polymerase is expressed,
and quickly begins to transcribe target gene which is then translated as desired protein. IPTG works
to displace a lac repressor since IPTG is an analogue of lactose. The lac genes express enzymes
which are involved in the breaking down of lactose, and therefore, the presence of lactose [or its
analogue] would trigger the initiation of transcription of lac genes.