Part:BBa_K3790214

T7lac::RBS::Bst-(G2S)3-S.ssb::K395601

Introduction

This composite part was used for IPTG induced Bst expression. A fusion version of the large fragment of Bst DNA polymerase I had replaced the native part K3790000.

Usage and Biology

T7 RNA Polymerase

The E. coli T7 phage has a specialized set of transcriptional elements, including promoters, ribosome-binding sites and terminators, and the expression system based on the use of those elements is called the T7 expression system. T7 RNA polymerase encoded by T7 phage gene 1 selectively activates transcription from a T7 phage promoter. It is a highly active RNA polymerase that synthesizes mRNA 5 times faster than E. coli RNA polymerase and can transcribe certain sequences that cannot be transcribed efficiently by E. coli RNA polymerase.

In the presence of T7 RNA polymerase and a T7 promoter in the bacteria, the transcription of the E. coli host's own genes greatly reduced. The transcription of genes that are controlled by the T7 phage promoter can reach very high levels.

T7 Expression Vectors

Based on these characteristics, expression vectors constructed with T7 phage gene elements have been available since the mid-1980s . Promoters were chosen over those of the T7 phage major coat protein sigma10 gene. The pET series of vectors is typical of this class of expression vectors, and vectors that have emerged later have all been developed on pET basis.

The pattern of transcriptional regulation by T7 RNA polymerase determines how the expression system is regulated. BL21 (DE3) was used as the expression host bacteria. The regulation is chemical signal inducible, similar to the lac expression. Only introducing the T7 RNA polymerase gene when needed, which reduce the background transcription to a very low level, especially for the expression of recombinant proteins those are toxic to E. coli.

Low Expression of T7 Lysozyme

The level of expression from the T7 system is the highest of all current expression systems. But still, it is inevitable that background transcription would affect bacterial growth if the gene product of interest is toxic to E. coli. One way to address this is to express the T7 lysozyme gene at low levels . Because T7 lysozyme, in addition to acting on peptidoglycan of the cell wall of Escherichia coli, binds to T7 RNA polymerase and inhibit its transcriptional activity.

All T7 lysozyme genes are introduced into the expression system by co-transformation plasmids, and it can reduce the background transcription, with no obvious effect on the expression level of target genes after IPTG induction.

T7 driven Bst expression

In our project, the T7 expression system (this part is based on the pET28 plasmid) was used to express the large fragment of Bst DNA polymerase I (large fragment), or its fusion.

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.

We designed this composite part to produce a fusion version of Bst polymerase (large fragment), trying to make the enzyme more efficient.

Experimental Results

Construction of K3700214

We obtained the Bst polymerase large fragment coding DNA optimized for Escherichia coli codon from Twist Bioscience, by total synthesis. We have the pET backbone in our laboratory stocks (namely pET16, pET28 and pET52; all of three shared the same backbone sequence used in our experiments).

File:T--Fudan--1002xxxxxxxxxxx.png

Figure 1. Gel electrophoresis of PCR produts of Bst fusions. 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 ClonExpress® Homologous Recombination Kit from Vazyme to perform PCR based cloning, placing the fusion Bst (Figure 1) between T7 RBS and T7 terminator. Homology arm primers used are documented at the Design page.

The Bst DNA polymerase I large fragment coding DNA is sized at 1764bp, and the (G2S)3-S.ssb fusion version is sized at 2235bp, which is approximately 2250bp after adding homology arms to both ends by PCR reaction. The result (Figure 1) shows a successful amplification of the DNA of interest. We performed gel extraction of the target region, to obtain pure Bst fusion with homology arms for subsequent reactions. Linearized pET backbone was obtained as described in K3790212.

After ClonExpress® homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight.

Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing.

After obtaining the correct K3790214 recombinant plasmids, we transformed them into BL21 (DE3) competent bacteria for protein expression. Positive colonies were selected by antibiotics preliminarily and then verified by colony PCR.

Bst Expression

The correct BL21 (DE3) colonies were picked into 2 mL antibiotics LB liquid medium overnight with shaking at 37 ℃.

The following day, the culture was transferred to 2 mL antibiotics LB broth at a 2% volume ratio, and incubation continued at 37 ℃ until reach the logarithmic growth phase of the bacteria (OD600 ~0.6). We then started IPTG induction, for at least 3 hours.

Table 1. Self-made BPCB (50 mL)

20 mmol/L Tris-HCl	25 mL, to a final concentration of 10 mmol/L
KCl	0.186 g, to a final concentration of 50 mmol/L
100 mmol/L DTT	0.5 mL, to a final concentration of 1 mmol/L
0.5 mol/L EDTA	10 μL, to a final concentration of 0.1 mmol/L
~24 mL ddH2O	Finalize to 50 mL
Triton X-100	v/v 0.1%
20 mmol/L Lysozyme (14 kDa)	Add 2 μL per 300 μL BPCB
PMSF	To a final concentration of 50 mmol/L

The induced bacterial culture were measure (OD600 absorbance, 1 OD equals to 10^8 950-nm diameter silica nanoparticles). Some of the bacterial culture was centrifuged to collect a pellet, and a self-made Bst polymerase compatible buffer (BPCB) was added (for formula, see Table 1). After lysis, we stored it at 4 ℃ before LAMP reaction. Another portion of the bacterial culture was directly centrifuged and the pellet was resuspended in 1x SDS sample buffer followed by immediate boiling at 100 ℃ for 5 minutes, will be used for whole-cell protein SDS-PAGE.

Bst fusions in BPCB for LAMP

File:T--Fudan--aBigLampDNA.png

Figure 2. LAMP reactions using self-made bacterial lysates, from Bst or Bst fusions. 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. All bacterial lysates were prepared after 3-hr IPTG induction.

Sequence and Features

Assembly Compatibility:

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