Overview

CountDown

Vistors From...

Characterization of our Favourite Parts

Abstract and Navigation

Part:BBa_K346000

Contents

• Part:BBa_K346000: main introduction

• Part:BBa_K346000: Part Design

• Part:BBa_K346000: 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.

[Top]

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)

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.

Part:BBa_K346001

Part:BBa_K346002

Part:BBa_K346003

Part:BBa_K346004

Part:BBa_K346005

Part:BBa_K346007