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Part:BBa_K346000

Contents

• Part:BBa_K346003: main introduction

• Part:BBa_K346003: Part Design

• Part:BBa_K346003: Experience

Part:BBa_K346000: main introduction

The RNA polymerase of bacteriophage T3 is DNA-directed RNA polymerase with both high template affinity and specificity. Similar to T7 polymerase, it also possesses quite simple structure compared with the bacterial RNA polymerase so that it can be easily wide-utilized in synthetic biology.

Previous work confirmed this concept and showed that although the RNAPs encoded by T3 and T7 bacteriophages have high sequence[1] , they exhibit nearly exclusive template specificities. According to the previous work, the two amino acid sequences have more than 82% identical residues[2]. Furthermore, promoters of the T7 and T3 RNAPs are also quite similar with highly conserved 23 bp sequences that differ significantly only over an about 3 bp region[2]. Despite these similarities, neither Polymerase (T3 RNA polymerase or T7 RNA polymerase) can transcribe the heterologous promoter efficiently[3,4] . Namely, the T3 RNA polymerase is orthogonal to T7 RNA polymerase, which is an excellent alternative of T7 polymerase for complex genetic circuit designed in the near future.

Figure 2. Predicted interaction between T3 polymerase and its cognate T3 promoter, based the data gained from T7 polymerase.

a. The interaction between bacterial RNA polymerase and the promoter DNA.

1. The -10 region interacts with the sigma factor;

2. The -35 region also interacts with the sigma factor;

3. The alpha CTD contacts the CRP protein.

b. The interaction between T3 polymerase and the T3 promoter.

1. The -1 to -4 TATA interacts with the 228 to 245 intercalating hairpin.

2. The -12 to -17 region of the promoter interacts with a beta loop of the T3 RNA polymerase.

3. The -5 to -12 region of the promoter interacts with the core specificity determinant.

Figure 3: The expression of T3 polymerase in BL21 strain. The strain bears a plasmid that has T7 promoter upstream of T3 polymerase. The left three columns represent strains that were not inducted by IPTG, and from left to right is protein expression in whole cell, cytosol, or inclusive body. The fourth column is the protein marker. The right three columns represent strains that were induced by 10^-3 M IPTG for 30 hours under 18℃. Note that there is a significant band in columns representing the induced strains as indicated by arrows, and the result shows that the T3 polymerase was mainly expressed in the cytosol.

Figure 4: The strength of different T3 promoters under the same expression level of T3 polymerase. BBa_E0240 was combined with 11 T3 promoters from the genome of T3 phage. The x axis denotes strains that only differ in the T3 promoter strength, in accordance with the order from strong to weak. The Y axis denotes the GFP intensity normalized by OD600. We can see that these T3 promoters can be divided into 3 subgroups: weak, medium and strong. (You can find more details about the promoter intensity characterization on the Experience Page)

Following is the key to the partsregistry number of each T3 promoter we characterized and their primary names in T3 phage genome.

Notice: phi13 has the same sequence with phi6.5.

It should be also noticed that as T3 RNA polymerase has dramatically high template affinity, even a little leakage expression of T3 polymerase might cause a significant basal level at T3 promoter. Therefore, when T3 polymerase is exploited in genetic circuit, its expression must be tightly controlled to reduce the basal level.

Reference

1. Davis, R. W. & Hyman, R. W (1971)J. Mol. Biol 62, 287-301.

2. McGraw, N. J., Bailey, J. N., Cleaves, G. R., Dembinski, D. R., Gocke, C. R., Joliffe, L. K., MacWright, R. S.&McAllister, W. T. (1985) Nucleic Acids Res. 13, 6753–6766.

3. Klement, J. F., Moorefield, M. B., Jorgensen, E. D., Brown, J. E., Risman, S. & McAllister, W. T. (1990) J. Mol. Biol. 215, 21–29.

4. Lee, S. S. & Kang, C. W. (1992) Biochem. Int. 26, 1–5.

5. Li, T., Ho, H. H., Maslak, M., Schick, C. & Martin, C. T. (1996) Biochemistry 35, 3722–3727.

6. Chapman, K. A. & Burgess, R. R. (1987) Nucleic Acids Res. 15, 5413–5432.

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Part:BBa_K346000: Part Design

T3 RNA polymerase has a dramatically high transcription rating; therefore a strong RBS is not necessary as a medium RBS (BBa_B0032) will be enough to reach the maximum of transcription activation at T3 promoters.

What’s more, we observed that maximum transcription rating might result in protein misfolding sometimes and the apparent transcription activation fold will decrease instead. Therefore we strongly recommend a low expression intensity of T3 polymerase in the case of genetic circuit design, or the combination of T3 polymerase with a low-strength T3 promoter, such as promoter phi 1.3 or 4.3. You can find more info about the T3 promoter intensity in the following Figure

Figure 5: The strength of different T3 promoters under the same expression level of T3 polymerase. BBa_E0840 was combined with 11 T3 promoters from the genome of T3 phage. The x axis denotes strains that only differ in the T3 promoter strength, in accordance with the order from strong to weak. The Y axis denotes the GFP intensity normalized by OD600. We can see that these T3 promoters can be divided into 3 subgroups: weak, medium and strong. (You can find more details about the promoter intensity characterization on the Experience Page)

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Part:BBa_K346000: Experience

We constructed a two-plasmid system to characterize the T3 polymerase and T3 promoters, using IPTG, as the inducer of T3 polymerase expression and GFP as the reporter gene.

Fig 1. The two-plasmid system we constructed to characterize T3 RNAP/T3 promoter pairs. T3 promoter was prefixed BBa_E0840 by insertion of annealing primers which would form EcoRI and SpeI sticky end. Induction temperature was 28-30℃

Induction time was 6-8h

IPTG concentration was 10^-3M; culture with no IPTG supplement as the negative control

Spin down the cells under 5000rpm, and discard the supernatant. Resuspend the pellet with PBS. The the intensity GFP was recorded by the microplate reader. Parameters were selected as suggested in BBa_E0840.

Figure 4: The strength of different T3 promoters under the same expression level of T3 polymerase (10^-3M IPTG). BBa_E0840 was combined with 11 T3 promoters from the genome of T3 phage. The x axis denotes strains that only differ in the T3 promoter strength, in accordance with the order from strong to weak. The Y axis denotes the GFP intensity normalized by OD600. We can see that these T3 promoters can be divided into 3 subgroups: weak, medium and strong.

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Part:BBa_K346001

Contents

• Part:BBa_K346001: main introduction

• Part:BBa_K346001: Part Design

• Part:BBa_K346001: Experience

Part:BBa_K346001: main introduction

Part:BBa_K346001: Part Design

Part:BBa_K346001: Experience

Part:BBa_K346002

Part:BBa_K346003

Designed by Huyang Tengxin Group: iGEM10_Peking (2010-10-14)

Contents

• Part:BBa_K346003: main introduction

• Part:BBa_K346003: Part Design

• Part:BBa_K346003: Experience

RBS(B0032)+MBP(mercury metal binding peptide engineered from MerR)

This part is a combination of a rbs, a mbp, the coding sequence of a mercury metal binding peptide egineered from MerR.It was designed to function as mercury(II) binding peptide in our project.It is expressed in the cytosol and when fused with the periplasm protein Dsba or the membrane protein lpp-ompa, it will be expressed and translocated to the periplasm and membrane.In short, this part function as the fundamental metal binding peptide of mercury in our project.

Part:BBa_K346003: main introduction

MerR, the mercury-responsible transcription factor(figure on the left), acts as an effective mercury accumulator in aquatic environment.However, as a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained and DNA binding domain and metal binding domain can function individually.Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) as is shown in the figure on the right.

The figure shows the structure of MerR and MBP. The left figure is MerR, the mercury-responsive transcription factor. The right figure shows the predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets.

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Part:BBa_K346000: Part Design

To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in the Fig. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step.Then the constructed mbp coding sequence is inserted to the plasmid

Source

The MerR is on the plasmid NR1, which is provided by Anne O. Summers.

References

Ralston, D. M. & Halloran, T. O. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. Proc. Natl. Acad. Sci. 87, 3846-3850 (1990).

Brocklehurst, K. R., Hobman, J. R., Lawley, B., Blank, L., Marshall, L. J., Brown, N. L. & Morby, A. P. ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli. Mol. Microbiol. 31, 893-902 (1999).

Zeng, Q., Stalhandske, C., Anderson, M. C., Scott, R. A. & Summers, A. O. The core metal-recognition domain of MerR. Biochemistry 37, 15885-15895 (1998).

Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., Halloran, T. V. & Mondrago, A. Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR. Science 301, 1383-1387 (2003).

Chen, P. R. & He, C. Selective recognition of metal ions by metalloregulatory proteins. Curr. Opin. Chem. Biol. 12,214-221 (2008).

Shewchuk, L. M., Verdine, G. L., Nash, H. & Walsh, C.T. Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription. Biochemistry 28, 6140-6145 (1989).

Wright, J. G., Tsang, H. T., Penner-Hahn, J. E. & O’Halloran T.V. Coordination chemistry of the Hg-MerR metalloregulatory protein: evidence for a novel tridentate Hg-cysteine receptor sites. J. Am. Chem. Soc. 112, 2434-2435 (1990).

Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., Halloran, T. V. & Mondrago, A. Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR. Science 301, 1383-1387 (2003).

Chen, P. R. & He, C. Selective recognition of metal ions by metalloregulatory proteins. Curr. Opin. Chem. Biol. 12,214-221 (2008).

Shewchuk, L. M., Verdine, G. L., Nash, H. & Walsh, C.T. Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription. Biochemistry 28, 6140-6145 (1989).

Wright, J. G., Tsang, H. T., Penner-Hahn, J. E. & O’Halloran T.V. Coordination chemistry of the Hg-MerR metalloregulatory protein: evidence for a novel tridentate Hg-cysteine receptor sites. J. Am. Chem. Soc. 112, 2434-2435 (1990).

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Part:BBa_K346003: Experience

To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix. The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig), which was verified by DNA sequencing.Then the RBS and terminator is added.

Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – mbp , which was verified by DNA sequencing.

Expression Experiment and Function Test:

To test the function of this part, both expression experiment and function test is necessary. We have verified the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES with the mercury gradient from 10^-8M to 10^-6M.

Results:

Expression of proteins

The plasmid PET21a-mbp is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD which consists with the predicted size, indicating that the engineered MBP can be expressed in the cytosol.

Function test Having made sure that the protein can express normally in the cytosol, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of MBP in different concentration of mercury, the concentration gradient is set from 10^-8M to 10^-6M, the results are shown in figure 3. It is obvious that the efficiency of MBP increases with the increase of the mercury concentration and it can binding Hg(II) with high efficiency and high sensitivity from the concentration of 10^-7 M compared to that of the control.

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Part:BBa_K346004

Part:BBa_K346005

Part:BBa_K346007