Part:BBa_K3790212

T7lac::RBS::Bst::K395601

Introduction

This composite part was used for IPTG induced Bst expression (without any fusion). The large fragment of Bst DNA polymerase I is critical for the isothermal amplification reaction in our project.

Usage and Biology

T7 expression system

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.

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.

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

Figure 1. Gel electrophoresis of PCR produts, using Twist synthesis as the template. 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. The arrowhead marks the target DNA band, approximately 1800bp.

In our project, the T7 expression system (based on the pET52 plasmid) was used to express the large fragment of Bst DNA polymerase I (large fragment), which is required for the isothermal amplification reaction (LAMP to be specific).

Our Bst (or previously called BstPol) 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

Construction of K3700212

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).

We used ClonExpress® Homologous Recombination Kit from Vazyme to perform PCR based cloning, placing 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, which is approximately 1800bp 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 with homology arms for subsequent reactions.

Figure 2. Gel electrophoresis of linearized pET bone. The first lane was loaded with DNA ladder, sizes were marked on the image. All bands are about 50 ng. The pET plasmid was subjected to loop PCR reaction with homology arm primers in order to linearize the vector. Clone16 and Clone52 were amplified from two different pET stock DNA templates. The arrowhead marks the target approximately 5200 bp.

Figure 2 indicates that we have obtained two copies of correct sized linearized pET backbone.

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

Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing (one correct sequencing result is shown in Figure 3).

Figure 3. Screenshot of one correct sequencing result, only small portion is shown. This clone was obtained using Clone52 linearized pET backbone. We sequenced the plasmids from both ends of Bst, and the valid sequences overlapped.

The plasmids containing sequencing verified K3790212 were restriction digested with NcoI and KpnI. The correct digestion pattern is shown in Figure 4.

Figure 4. Gel electrophoresis of plasmids containing K3790212 digested with NcoI and KpnI. The first lane was loaded with DNA ladder, sizes were marked on the image. All bands are about 50 ng. Lane labeled with K3790212 is the plasmid DNA without enzymatic digestion. Lane labeled with NcoI/KpnI, is digested. The arrowhead marks the predicted excised DNA fragment approximately 1800bp.

Figure 5. Gel electrophoresis of colony PCR of BL21 (DE3) clones. 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. After obtaining the correct K3790212 recombinant plasmids, we transformed them into BL21 (DE3) competent bacteria for protein expression. Positive colonies were selected by ampicillin preliminarily and then verified by colony PCR. The arrowhead marks the target approximately 1800bp.

Bst Expression

Figure 6. 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.

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

The following day, the culture was transferred to 2 ml ampicillin 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.

The induced bacterial culture were measure (OD600 absorbance). 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 and 4 ℃ 13000rpm 10-min centrifugation, we collected the supernatant (containing Bst polymerase, Figure 6), to perform 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 protein SDS-PAGE (Figure 8).

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

Bst in BPCB for LAMP

The two lanes in Figure 7 show that Candida albicans DNA strongly amplified by a lysate of K3790212 containing, IPTG induced, BL21 (DE3) bacteria. The specificity of DNA product was later verified with lateral flow assay (LFA).

Figure 7. 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.

The initial test with our gp5.7 plasmid

For more details about our gp5.7 plasmids, please visit https://parts.igem.org/Part:BBa_K3790232

Figure 8. Compare whole bacteria protein under different induction condition. BL21 (DE3) bacteria, transformed with either K3790212 alone, or with both K3790212 and K3790232, were cultured to OD600 ~0.6 and then induced as indicated for 3 hours. Whole cell lysates were prepared by directly adding 1x SDS sample buffer into bacteria pellets, followed by immediate boiling at 100 ℃ for 5 minutes. Lane 5 and Figure 6, was made using a same culture, IPTG induced for 3 hours.

Sequence and Features

Assembly Compatibility:

• 10
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• 12
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• 21
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• 23
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• 25
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• 1000
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