Difference between revisions of "Part:BBa K4907107"
 
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This composite part was designed to express RFP, which are assembled on the expression vector pSB3K3 by standard assembly. To verify the orthogonality of T7 RNA polymerase and pVSW-3 (18) RNA polymerase, the constructed plasmids and plasmid <partinfo>BBa_K4907114</partinfo>_pSB1C3 were transformed into ''E. coli'' DH10β. Then the positive transformants were selected by kanamycin and chloramphenicol and confirmed by colony PCR and sequencing. After being induced by L-arabinose, pVSW-3 (18) RNA polymerase will be produced. Then the intensity of red fluorescence was detected to figure out whether T7 promoter can be activated by pVSW-3 (18) RNA polymerase.
 
This composite part was designed to express RFP, which are assembled on the expression vector pSB3K3 by standard assembly. To verify the orthogonality of T7 RNA polymerase and pVSW-3 (18) RNA polymerase, the constructed plasmids and plasmid <partinfo>BBa_K4907114</partinfo>_pSB1C3 were transformed into ''E. coli'' DH10β. Then the positive transformants were selected by kanamycin and chloramphenicol and confirmed by colony PCR and sequencing. After being induced by L-arabinose, pVSW-3 (18) RNA polymerase will be produced. Then the intensity of red fluorescence was detected to figure out whether T7 promoter can be activated by pVSW-3 (18) RNA polymerase.
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/csq/t7-promoter-b0034-rfp-b0015.png" width="400px"></html></center>
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<center><html><B>Fig. 1 Gene circuit of BBa_K4907107.</B></html></center>
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===Characterization===
 
===Characterization===
  
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/csq/bba-k4907107-t7-promoter-b0034-rfp-b0015-csq111-1.png" width="400px"></html></center>
 
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/csq/bba-k4907107-t7-promoter-b0034-rfp-b0015-csq111-1.png" width="400px"></html></center>
  
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<B>Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907107_pSB3K3.</B>
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<center><html><B>Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907107_pSB3K3.</B></html></center>
  
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====Double transformation====
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====Dual-plasmid system====
 
Plasmid <partinfo>BBa_K4907107</partinfo>_pSB3K3 and plasmid <partinfo>BBa_K4907114</partinfo>_pSB1C3 were transformed into ''E. coli'' DH10β. The positive transformants were selected by kanamycin and chloramphenicol.
 
Plasmid <partinfo>BBa_K4907107</partinfo>_pSB3K3 and plasmid <partinfo>BBa_K4907114</partinfo>_pSB1C3 were transformed into ''E. coli'' DH10β. The positive transformants were selected by kanamycin and chloramphenicol.
 
====Fluorescence measurement ====
 
====Fluorescence measurement ====
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Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of RFP is observed by measuring the Fluorescence/OD<sub>600</sub> using microplate reader (Fig. 2). The results of fluorescence showed that T7 promoter cannot be activated by pVSW-3 (18) RNA polymerase and pVSW-3 (18) also cannot be activated by T7 RNA polymerase. The above experimental results indicate that the VSW-3 expression system itself is relatively rigorous in ''E. coli'', and the T7 expression system is orthogonal to the VSW-3 expression system. So we could use the VSW-3 expression system for gate design, and even the design of new specific expression bacteria and corresponding vectors.
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Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of RFP is observed by measuring the Fluorescence/OD<sub>600</sub> using microplate reader. The results of fluorescence showed that T7 promoter cannot be activated by pVSW-3 (18) RNA polymerase (Fig. 2a) and pVSW-3 (18) also cannot be activated by T7 RNA polymerase (Fig. 2b). The above experimental results indicate that the VSW-3 expression system itself is relatively rigorous in ''E. coli'', and the T7 expression system is orthogonal to the VSW-3 expression system. So we could use the VSW-3 expression system for gate design, and even the design of new specific expression bacteria and corresponding vectors.
  
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<B>Fig. 2 The result of the ratio of red fluorescence to OD<sub>600</sub></B>.
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/csq/iptg-ara111.png" width="700px"></html></center>
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<center><html><B>Fig. 3 The result of the ratio of red fluorescence to OD<sub>600</sub></B>.</html></center>
  
 
====P<sub>T7</sub> temperature gradient experiment====
 
====P<sub>T7</sub> temperature gradient experiment====
 
We transferred <partinfo>BBa_K4907107</partinfo>_pSB3K3 into ''E. coli'' BL21(DE3), induced by IPTG, and performed kinetic experiments at 30 ℃, 25 ℃, and 15 ℃, respectively. As shown in the figure (Fig. 3), the expression efficiency of <partinfo>BBa_K4907107</partinfo>_pSB3K3 decreased with the decrease of temperature.
 
We transferred <partinfo>BBa_K4907107</partinfo>_pSB3K3 into ''E. coli'' BL21(DE3), induced by IPTG, and performed kinetic experiments at 30 ℃, 25 ℃, and 15 ℃, respectively. As shown in the figure (Fig. 3), the expression efficiency of <partinfo>BBa_K4907107</partinfo>_pSB3K3 decreased with the decrease of temperature.
  
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<B>Fig. 3 The ratio of red fluorescence to OD<sub>600</sub> changes with the change of temperature</B>.
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/csq/t7wendutidu.png" width="400px"></html></center>
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<center><html><B>Fig. 4 The ratio of red fluorescence to OD<sub>600</sub> changes with the change of temperature</B>.</html></center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:26, 10 October 2023


T7 promoter-B0034-rfp-B0015

Biology

RFP

A gene codes for the red fluorescence protein which is used as a marker. The expression level of RFP can be used to characterize the function of promoters.

T7 promoter

T7 promoter is specifically controlled by the T7 RNA polymerase.

Usage and design

This composite part was designed to express RFP, which are assembled on the expression vector pSB3K3 by standard assembly. To verify the orthogonality of T7 RNA polymerase and pVSW-3 (18) RNA polymerase, the constructed plasmids and plasmid BBa_K4907114_pSB1C3 were transformed into E. coli DH10β. Then the positive transformants were selected by kanamycin and chloramphenicol and confirmed by colony PCR and sequencing. After being induced by L-arabinose, pVSW-3 (18) RNA polymerase will be produced. Then the intensity of red fluorescence was detected to figure out whether T7 promoter can be activated by pVSW-3 (18) RNA polymerase.


Fig. 1 Gene circuit of BBa_K4907107.

Characterization

Identification

After constructing this circuit, colony PCR and gene sequencing were used to verify that the plasmid was correct. Target bands (1188 bp) could be observed at the position around 1000 bp (Fig. 1).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907107_pSB3K3.

Dual-plasmid system

Plasmid BBa_K4907107_pSB3K3 and plasmid BBa_K4907114_pSB1C3 were transformed into E. coli DH10β. The positive transformants were selected by kanamycin and chloramphenicol.

Fluorescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of RFP is observed by measuring the Fluorescence/OD600 using microplate reader. The results of fluorescence showed that T7 promoter cannot be activated by pVSW-3 (18) RNA polymerase (Fig. 2a) and pVSW-3 (18) also cannot be activated by T7 RNA polymerase (Fig. 2b). The above experimental results indicate that the VSW-3 expression system itself is relatively rigorous in E. coli, and the T7 expression system is orthogonal to the VSW-3 expression system. So we could use the VSW-3 expression system for gate design, and even the design of new specific expression bacteria and corresponding vectors.

Fig. 3 The result of the ratio of red fluorescence to OD600.

PT7 temperature gradient experiment

We transferred BBa_K4907107_pSB3K3 into E. coli BL21(DE3), induced by IPTG, and performed kinetic experiments at 30 ℃, 25 ℃, and 15 ℃, respectively. As shown in the figure (Fig. 3), the expression efficiency of BBa_K4907107_pSB3K3 decreased with the decrease of temperature.

Fig. 4 The ratio of red fluorescence to OD600 changes with the change of temperature.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 473
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 595
  • 1000
    COMPATIBLE WITH RFC[1000]