Experiment: We selected 8 different promoters for our chemoreceptor TAR. We chose the following parts for this purpose:
J23100 (18C) J23104 (18K) J23105 (18M) J23106 (18O) J23109 (2G) J23112 (20E) J23113 (20G) J23114 (20I) all of these in vector J61002 (for details click here). These constructs were, prior to this experiment, transformed into E. coli, prepped and stored at -20 °C.
The vector J61002 was digested with SpeI and PstI, the insert TAR_QC was digested with XbaI and PstI and subsequently ligated downstream of our 8 promoters according to protocol. We transformed E. coli Δ tar with the generated constructs. This is an E. coli DH10B strain where the tar gene is deleted. Consequently this strain cannot perceive Aspartate any longer. We use it to verify that our constructs are able to rescue this phenotype.
V06_13
Preparation of J61002_promoter_TAR_QC
Experiment: All 8 promoter constructs + TAR_QC were prepped using peqGOLD Plasmid Miniprep kit (PeqLab) following the user manual. We performed a test digestion using XbaI and PstI to verify that the insert TAR_QC was successfully integrated into the plasmid. Glycerol stocks (30 %) of E. coli Δ tar J61002_promoter_TAR_QC were prepared and stored at -80 °C.
Observations & Results: All eight constructs were successfully generated!
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