Part:BBa_K346000

RBS(B0032)+T3 DNA-directed RNA polymerase

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.

FIG.1 The predicted structure based on the data of T7 polymerase

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 parts.igem number of each T3 promoter we characterized and their primary names in T3 phage genome.

phiOL	[BBa_K346039];	phiOR	[BBa_K346040];	phi1.05	[BBa_K346041];	phi1.1	[BBa_K346042];	phi1.3	[BBa_K346043];
phi1.5	[BBa_K346044];	phi2.5	[BBa_K346045];	phi4.3	[BBa_K346046];	phi6.5	[BBa_K346047];	phi9	[BBa_K346048];
phi10	[BBa_K346049];	phi11	[BBa_K346050];	phi13	[BBa_K346051];	phi3.8	[BBa_K346052];	-	-

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2585

Functional Parameters

biology	Orthogonal to T7 polymerase/T7 promoter pair
family	Phage and mitochondrial RNA polymerase family
function	DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates.
n/a	RBS(B0032)+T3 DNA-directed RNA polymerase
negative_regulators	Bacterial RNA polymerase inhibitor from T3 phage
origin	Enterobacteria phage T3 (Bacteriophage T3)
rbs	Medium
uniprot	P07659

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.