Part:BBa_K3790000

Bst polymerase I (large fragment) for E. coli

This part is improved from BstPol https://parts.igem.org/Part:BBa_K3416003

Introduction

This basic part, as an improved version of K3416003, is the coding sequence of the large fragment of Bst DNA polymerase I, which is critical for the isothermal amplification reaction in our project.

Usage and Biology

In our project, K3790000 is the gene of interest in the T7 expression system. The large fragment of Bst DNA polymerase I is required for the isothermal amplification reaction (LAMP to be specific).

Our Bst is derived from the DNA polymerase I large fragment in Bacillus stearothermophilus. The large fragment of Bst DNA Polymerase I contains 5' to 3' DNA polymerase activity and strong strand displacement activity, but lacks 5' to 3' exonuclease activity, ideal for target sequence amplification.

Experimental Results

Figure 1. Amplified Bst polymerase I (large fragment). The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. We used Twist Bioscience synthesized DNA as PCR template, which was about 1-month waiting after we sent in the sequence. After PCR cloning into desired plasmids, several bacterial clones were picked, grew into cultures and sent for Sanger sequencing, from both ends (the sequencing results from both ends must overlap). Then, we used the correct ones for further experiments.

The length of Bst DNA is approximately 1800bp after adding homology arms to both ends for PCR cloning (Figure 1). We isolated the DNA of interest by gel extraction for subsequent reactions.

Figure 2. Compare proteins in the pellet vs. supernatant, after BPCB lysis. Lane 2 was Bst containing supernant. We add equal volume of 2x SDS sample buffer. Lane 1 was BPCB insolvable components, from equivalent pellet. As shown in the gel, BPCB solubilizes the bacteria very well, and we do not perform 4 ℃ centrifugation after seeing this gel.

After constructing recombinant bacteria and inducing expression, we successfully obtained bacterial lysate containing the large fragment of Bst polymerase with a self-made Bst polymerase compatible buffer (BPCB) lysis (Figure 2) and confirmed the DNA amplification activity of the lysate in a LAMP reaction (Figure 3), whose specificity was later verified with lateral flow assay (LFA).

Figure 3. LAMP reactions using purified Bst or self-made bacterial lysates. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. For the last two lanes, we used bacterial lysates from BL21 (DE3) transformed with K37900212, with or without IPTG induction. Purified Bst represents the purified enzyme.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]