Part:BBa_K3416003
BstPol
Introduction
Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This Bst polymerase I large fragment (BstPol) is a part of helimerase protein complex, which could be used with the aim to improve HDA method.
Contents
Biology
Since DNA polymerases are one of the main molecular biology tools, they are mostly used in different amplification methods such as PCR, RPA, LAMP or HDA[1]. One type of polymerase, which can be used in these methods, is thermostable DNA polymerase I large fragment (EC: 2.7.7.7.), obtained from Bacillus stearothermophilus. This enzyme (67.7 kDa) has a strand displacement and 5’-3’ exonuclease activity. Based on scientific research, the best suitable polymerase activity temperature varies from 65 °C to 72 °C[2].
In project FlavoFlow we a novel isothermal helicase dependent amplification method, which is based on DNA replication fork principle. Usually, in this assay, two complementary DNA strands are being separated with UvrD helicase. When newly generated ssDNA is being coated with single-stranded DNA-binding proteins. The next step of HDA process is the hybridization of site-specific primers to each ending border of ssDNA. After this hybridization the elongation process performed by DNA polymerase, starts[1]. The biggest advantage of this HDA reaction is its specificity as well as the ability to perform the reaction at a constant temperature.
However, even if HDA method is sensitive and specific, it is able to amplify shorter than 200 bp sequences[3]. This problem was solved by fusing TteUvrD helicase (BBa_K3416002) with Bst polymerase I large fragment (BstPol) through coiled-coil interaction (Fig. 1). Such fusion increases the speed and synthesis of new strands, which makes HDA reaction more specific and efficient. Based on similar researches, scientists have shown, that coordination between TteUvrD helicase and BstPol could increase specificity and efficiency of the HDA reaction[3],[4]. Previously mentioned proteins BstPol and TteUvrD are physically linked together by using coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is possessed in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provides predictable tertiary structure and stability[5].
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Figure 1. Bifunctional protein helimerase. TteUvrD - thermostable UvrD helicase, BstPol - Bst polymerase I large fragment, L1 - linker between WZB1/WZA2 coiled-coil amino acid structures, WZA2 - coiled-coil amino acid structure, fused with TteUvrD, WZB1 - coiled-coil amino acid structure, fused with BstPol, 10xHisTag - TteUvrD purification tag, Maltose binding protein (MBP) - TteUvrD purification tag, StrepII tag - BstPol purification tag.
The most important feature of these coiled-coil amino acid sequences is a seven-residue repeat (abcdefg)n, where first (a) and fourth (d) positions need to be occupied by hydrophobic amino acids by the means to determine the oligomerization state. In parallel residues at seventh (g) and the succeeding fifth (e) positions forms hydrogen-bonded ionic interactions[4]. In our case, these coiled coils were constructed based on c-Jun/c-Fos and GCN4 Leucine zippers, where parallel dimer is formed with the leucine in d sites and valine in a sites [3]. According to the research, the most stable heterodimer is formed with a TM of 63 °C, where binding affinity constant reached Kd of 4.5 nM[3].
Results
Helimerase – is a bifunctional protein complex, made up of two enzymes - BstPol and TteUvrD. These proteins are physically linked to each other through the coiled-coil structure. TteUvrD helicase is fused with one part of this structure, WinZip-A2 (WZA2), through the linker L1 and is possessed in the N-terminal end of the sequence. The research has shown that this type of coiled-coils structures provides predictable tertiary structure and stability[3]. Firstly gene sequence WZA2-L1-TteUvrD was cloned into pETDuet vector. This protein was fused with a 10xHis tag and a maltose binding protein (MBP) at N terminus. Analogously, WZB1-L1-BstPol gene was cloned into the second MCS of pACYC plasmid and fused with StrepII tag at the N terminus.
The optimal expression conditions for WZB1-L1-BstPol was observed in E. coli BL21(DE3) strain after 16 hours induction with 1 mM IPTG at 30 °C. Protein was purified under native conditions. WZB1-L1-BstPol protein was purified by using Ni-NTA affinity column. The yield of WZB1-L1-BstPol obtained after purification from 1 L medium was 2.2 mg/ml (Fig. 2)
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Figure 2. SDS-PAGE electrophoresis after WZA2-L1-TteUvrD (136.598 kDa) and WZB1-L1-BstPol (74.225 kDa) purification. L - PageRuler Plus Prestained Protein Ladder (#26620), 1 - WZA2-L1-TteUvrD supernatant, 2 - WZA2-L1-TteUvrD 3 elution fraction, 3 - WZA2-L1-TteUvrD 5 elution fraction, 4 - WZA2-L1-TteUvrD 14 elution fraction, 5 - WZA2-L1-TteUvrD after protein concentration, 6 - WZB1-L1-BstPol supernatant, 7 - WZB1-L1-BstPol 4 elution fraction, 8 - WZB1-L1-BstPol 6 elution fraction, 9 - WZB1-L1-BstPol 7 elution fraction, 10 - WZB1-L1-BstPol after protein concentration. SDS-PAGE electrophoresis after WZA2-L1-TteUvrD (136.598 kDa) and WZB1-L1-BstPol (74.225 kDa) purification. L - PageRuler Plus Prestained Protein Ladder (#26620), 1 - WZA2-L1-TteUvrD supernatant, 2 - WZA2-L1-TteUvrD 3 elution fraction, 3 - WZA2-L1-TteUvrD 5 elution fraction, 4 - WZA2-L1-TteUvrD 14 elution fraction, 5 - WZA2-L1-TteUvrD after protein concentration, 6 - WZB1-L1-BstPol supernatant, 7 - WZB1-L1-BstPol 4 elution fraction, 8 - WZB1-L1-BstPol 6 elution fraction, 9 - WZB1-L1-BstPol 7 elution fraction, 10 - WZB1-L1-BstPol after protein concentration.
To verify that WZB1-L1-BstPol protein is stable at high temperature we performed fluorescence thermal shift assay by utilizing a thermodynamic model3. This measurement is based on protein property to unfold upon increasing temperature of environment and solvatochromic property of fluorescence dye to fluorescent more intensively in the hydrophobic environment. The results (Fig. 3) show that protein is stable at high temperatures, thus letting us use it in activity assays. Also, according to obtained melting temperatures (TM) WZB1-L1-BstPol enzyme is stable and starts to denature at 76 °C.
WZB1-L1-BstPol activity (Fig. 4A and Fig. 4B) was also measured by observing its fluorescence depending on time. The bigger polymerase concentration is in the assay, the more amplified fragments are being generated. Linear dependence of polymerase activity on its substrate in solution was also determined by using fluorescence measurements.
WZB1-L1-BstPol activity (Fig. 5A and Fig.5B) was also measured by observing its fluorescence depending on time. The bigger polymerase concentration is in the assay, the more amplified fragments are being generated. Linear dependence of polymerase activity on its substrate in solution was also determined by using fluorescence measurements.
Based on these enzymes activity assays we determined that the synthesis of active helimerase proteins was successful. However, after a lot of attempts we were unable to fuse these proteins via coiled-coil amino acid structures in vivo and in vitro. We hypothesized that newly formed affinity tags, as such as tertiary structures, could influence these proteins' interaction with each other, so in order to tackle this problem, new affinity tags and linkers should be tested. Also, with the aim to co-express these proteins in vivo, they could be cloned into other compatible plasmids.
Added by 2021 Fudan
This part's sequence contains a STOP
The last 39 nucleotides in K3416003, as: gcg aag tga cttgagcaccatcat cctaggcaccaccac-3 which translate into: AK* LEHHH PRHHH The STOP codon (tga) was not a mistake, but an intention. Please note the purification was using Strep Tag II but not His10.
Amino-acid sequences alignment
We remove the heading Strep Tag II (used for purification) and WZB1-L1 (used for heterodimerization).
If you are looking for a native Bst polymerase I large fragment, please visit https://parts.igem.org/Part:BBa_K3790000
K3416003 RGSHMKSSWSHPQFEKAVSSGLVPRGSHMVDELQAEVDQLQDENYALKTKVAQLRKKVEK
K3790000 ------------------------------------------------------------
K3416003 LGAAVLVGAAVLVAGAGAAVGAEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYH
K3790000 --------------------MAEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYH
***************************************
K3416003 DAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELR
K3790000 DAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELR
************************************************************
K3416003 GVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLV
K3790000 GVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLV
************************************************************
K3416003 RKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELA
K3790000 RKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELA
************************************************************
K3416003 EQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHH
K3790000 EQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHH
************************************************************
K3416003 EIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPI
K3790000 EIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPI
************************************************************
K3416003 RLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMD
K3790000 RLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMD
************************************************************
K3416003 IFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQ
K3790000 IFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQ
************************************************************
K3416003 YMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAM
K3790000 YMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAM
************************************************************
K3416003 IDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHY
K3790000 IDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHY
************************************************************
K3416003 GPTWYDAK*LEHHHPRHHH
K3790000 GPTWYDAK-----------
********
Codon optimized for bacterial expression
K3790000 has been codon optimized for induced expression in E. coli.
Please check detailed nucleotide sequence at https://parts.igem.org/Part:BBa_K3790000
Use bacteria lysate for isothermal DNA amplification
As described at https://2021.igem.org/Team:Fudan/Design the testing schema developed by 2021 Fudan is for resource-limited regions. There are no equipment for protein purification, nor reliable low-temperature transportation routes to those areas.
As shown at https://2021.igem.org/Team:Fudan/Results
- BstPol in bacterial lysate could be used directly for DNA amplification, with comparable outcome as purified proteins
- Inspired by https://2017.igem.org/Team:TUDelft/Demonstrate bacterial lysate could be prepared with easily accessible tools, although chemical used need to be shipped as dry powder
- The purity of BstPol in the lysate has been improved, using a general gene expression suppression circuit, more details at https://parts.igem.org/Part:BBa_K3790050 and https://parts.igem.org/Part:BBa_K3790085
Fuse DNA binding domains with BstPol
We improved BstPol with fusions (Bst = BstPol; DbpA, S.ssb, E.ssb, albA1, Sso7d, Sso10b are different DNA binding domains). More details at https://2021.igem.org/Team:Fudan/Parts
- Bst-(G2S)3-DbpA https://parts.igem.org/Part:BBa_K3790150
- DbpA-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790156
- Bst-(G2S)3-S.ssb https://parts.igem.org/Part:BBa_K3790151
- S.ssb-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790157
- Bst-(G2S)3-E.ssb https://parts.igem.org/Part:BBa_K3790152
- E.ssb-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790162
- Bst-(G2S)3-albA1 https://parts.igem.org/Part:BBa_K3790153
- albA1-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790159
- Bst-(G2S)3-Sso7d https://parts.igem.org/Part:BBa_K3790154
- Sso7d-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790161
- Bst-(G2S)3-Sso10b https://parts.igem.org/Part:BBa_K3790155
- Sso10b-(G2S)3-Bst https://parts.igem.org/Part:BBa_K3790160
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ 1.0 1.1 Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Reports 5, 795–800 (2004).
- ↑ Kiefer, J. R. et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 Å resolution. Structure 5, 95–108 (1997).
- ↑ 3.0 3.1 3.2 3.3 3.4 Motré, A., Li, Y. & Kong, H. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase. Gene 420, 17–22 (2008).
- ↑ 4.0 4.1 Muller, K.M., Arndt, K.M., Albert, T. Protein fusions to coiled-coil domains. Methods Enzymol. 328, 261-282.
- ↑ Cimmperman, P. et al. A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophysical Journal 95, 3222–3231 (2008).
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