After successful PCR on extracted E. coli RNA and determination of HNB and MgSO4 concentrations, LAMP was carried out on E. coli RNA and genomic DNA from the same strain. Amplified samples were run on an agarose gel to observe activity which confirmed successful design of all LAMP primers and strong activity of reverse transcriptase on RNA samples (Fig. 1).

Figure 1. LAMP reaction for 3 samples of E. coli K-12 RNA and 3 samples of genomic DNA. High mass of all positive samples indicates successful reaction and activity from all LAMP components.

The pET24d vector was used for cloning modified Bst DNA polymerase fusion with DNA binding protein Sac7e due to the inclusion of a 6x histidine tag, as well as BamHI and XhoI restriction enzymes positioned to amplify our construct in the correct direction. Plasmid designed using SnapGene software.

Figure 2. Plasmid map of Bst_mod_Sac7e inserted into pET24d. 6x His tag and multiple cloning site (MCS) highlighted in purple and blue, respectively.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) used to induce protein expression (1). Modified Bst constructs with fusion with Sso7d (Fig. 3a) and Sac7e (Fig. 3b) binding proteins were both expressed and then purified through nickel chromatography. Both constructs were successfully expressed, however only Bst_mod_Sso7d was seen after purification. A Western blot confirmed these observations as well.

Figure 3. SDS-PAGE gels for Bst_mod_Sso7d (a) and Bst_mod_Sac7e (b) after expression and purification. Crude, supernatant, pellet, and elution samples for both uninduced and IPTG-induced proteins are displayed. Presence of a band at 74.6 kDa indicates successful purification of Bst_mod_Sso7d.

All trials and associated results can be found in our Lab Notebook.