Difference between revisions of "Part:BBa K346000"
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<partinfo>BBa_K346000 short</partinfo>
 
<partinfo>BBa_K346000 short</partinfo>
  
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T3 polymerase can activate the expression of different T3 promoters in T3 phage. Since during different time the phage need different expression level of different genes, there must be T3 promoters of variable sensitivity.
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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.
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In our experiment, we constructed 14 different T3 promoter by primer annealing and by standard digestion to put it into standard plasmid. We then add GFP downstream to function as a reporter. By transforming another plasmid contains T7 promoter+RBS+T3 polymerase, we were able to detect the expression level of different promoters. We finally constructed 9 promoters with variable sensitivity, and put them in order according to the GFP expression level. We found that among them the Phi9 T3 promoter has a medium expression level and was more robust in the system, so we chose this one to be used in our kit.
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<!-- Add more about the biology of this part here
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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[3]. Despite these similarities, neither Polymerase (T3 RNA polymerase or T7 RNA polymerase) can transcribe the heterologous promoter efficiently[4,5] . 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.  
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===Usage and Biology===
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<html><img src="https://static.igem.org/mediawiki/parts/1/14/T3POLfig1.png" width=600 target="blank" ></html>
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<html><img src="https://static.igem.org/mediawiki/parts/6/6a/T3POLfig2.png" width=600 target="blank"></html>
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'''Figure 2. Predicted interaction between T3 polymerase and its cognate T3 promoter, based the data gained from T7 polymerase.'''
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a. The interaction between bacterial RNA polymerase and the promoter DNA.
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1. The -10 region interacts with the sigma factor;
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 +
2. The -35 region also interacts with the sigma factor;
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3. The alpha CTD contacts the CRP protein.
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b. The interaction between T3 polymerase and the T3 promoter.
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1. The -1 to -4 TATA interacts with the 228 to 245 intercalating hairpin.  
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2. The -12 to -17 region of the promoter interacts with a beta loop of the T3 RNA polymerase.  
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3. The -5 to -12 region of the promoter interacts with the core specificity determinant.
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<html><img src="https://static.igem.org/mediawiki/parts/7/76/T3POLfig3.png" width="400" target="blank"></html>
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'''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.
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<html><img src="https://static.igem.org/mediawiki/parts/a/ae/T3POLfig4.png" target="blank"></html>
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'''Figure 4: 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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Following is the key to the parts.igem number of each T3 promoter we characterized and their primary names in T3 phage genome.
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{| class="name" border="2" rules="rows" width="650px" style="color:#000000;background-color:#ffff00"
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|-
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|style="text-align:center"| phiOL
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346039 BBa_K346039]];
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|style="text-align:center"| phiOR
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346040 BBa_K346040]];
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|style="text-align:center"| phi1.05
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346041 BBa_K346041]];
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|style="text-align:center"| phi1.1
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346042 BBa_K346042]];
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|style="text-align:center"| phi1.3
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346043 BBa_K346043]];
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|-
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|style="text-align:center"| phi1.5
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346044 BBa_K346044]];
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|style="text-align:center"| phi2.5
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346045 BBa_K346045]];
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|style="text-align:center"| phi4.3
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346046 BBa_K346046]];
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|style="text-align:center"| phi6.5
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346047 BBa_K346047]];
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|style="text-align:center"| phi9
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346048 BBa_K346048]];
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|-
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|style="text-align:center"| phi10
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346049 BBa_K346049]];
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|style="text-align:center"| phi11
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346050 BBa_K346050]];
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|style="text-align:center"| phi13
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346051 BBa_K346051]];
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|style="text-align:center"| phi3.8
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|style="text-align:center"| [[https://parts.igem.org/Part:BBa_K346052 BBa_K346052]];
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|style="text-align:center"| -
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|style="text-align:center"| -
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|}
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Notice:  phi13 has the same sequence with phi6.5.
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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.
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==Reference==
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1. Davis, R. W. & Hyman, R. W (1971)J. Mol. Biol 62, 287-301.
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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.
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3. 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.
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 +
4. Klement, J. F., Moorefield, M. B., Jorgensen, E. D., Brown, J. E., Risman, S. & McAllister, W. T. (1990) J. Mol. Biol. 215, 21–29.
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5. Lee, S. S. & Kang, C. W. (1992) Biochem. Int. 26, 1–5.
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Revision as of 07:00, 25 October 2010

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[3]. Despite these similarities, neither Polymerase (T3 RNA polymerase or T7 RNA polymerase) can transcribe the heterologous promoter efficiently[4,5] . 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_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)


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. 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.

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

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


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