Difference between revisions of "Part:BBa K3866001"
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The trc promoter is a hybrid of the lac and trp promoters which is a stronger promoter in comparison to the lac promoter.
 
The trc promoter is a hybrid of the lac and trp promoters which is a stronger promoter in comparison to the lac promoter.
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[[Image:T--Thessaly--ptrcsnap.png|800px|thumb|none|<I><b>Figure 1.</b> The level 0 module : pupd2- pTrc:RBS (illustration from SnapGene)</i>]]
  
 
===Usage and Biology===
 
===Usage and Biology===
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The trc promoter was created by insertion of 1 bp into the 16 bp sequence between the consensus -35 and -10 sequences of the tac promoter, to obtain the optimal consensus distance of 17 bp between the -35 and -10 signal. This promoter includes the <i>Lac Operator</i> sequence lacO, which can be bound by the Lac repressor lacI, as well as an RBS <bbpart>BBa_B0034</bbpart>. For inducible gene expression from the trc promoter the lac repressor (LacI) is necessary. The repressor binds to the operator DNA with high specificity and inhibits gene expression until an inducer like allolactose or the analog IPTG (Isopropyl-?-D-thio-galactoside) is added to the medium. The inducer binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNA polymerase (RNAP) to transcribe the following genes and thereby leading to high levels of the encoded proteins.
 
The trc promoter was created by insertion of 1 bp into the 16 bp sequence between the consensus -35 and -10 sequences of the tac promoter, to obtain the optimal consensus distance of 17 bp between the -35 and -10 signal. This promoter includes the <i>Lac Operator</i> sequence lacO, which can be bound by the Lac repressor lacI, as well as an RBS <bbpart>BBa_B0034</bbpart>. For inducible gene expression from the trc promoter the lac repressor (LacI) is necessary. The repressor binds to the operator DNA with high specificity and inhibits gene expression until an inducer like allolactose or the analog IPTG (Isopropyl-?-D-thio-galactoside) is added to the medium. The inducer binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNA polymerase (RNAP) to transcribe the following genes and thereby leading to high levels of the encoded proteins.
  
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===Design Notes===
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<span class='h3bb'>Sequence and Features</span>
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The sequence was domesticated. We removed BsmBI, BsaI, BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 <bbpart>BBa_K3505007</bbpart> and has overhangs compatible for Golden Braid cloning.
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<partinfo>BBa_K3866001 SequenceAndFeatures</partinfo>
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This has position A1-B2.
  
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[[File:T--Thessaly--GB-GGAG-AATG.jpeg|800px|thumb|none|<i><b>Figure 1.</b>The overhangs of this part in the GoldenBraid Grammar</i>]]
  
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===Verification of cloning===
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===Functional Parameters===
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<partinfo>BBa_K3866001 parameters</partinfo>
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[[File:T--Thessaly--pTrc pLac--digestion.png|500px|thumb|none|<i><b>Figure 3.</b> (C= Cut, U=Uncut) Restriction digestion of pTrc:RBS (C1 + C2) with: EcoRI + EcoRV, Expected bands: 1280bp, 896bp, Positive result: C1 + C2</i>]]
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===Source===
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Synthesized by IDT.
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===Sequence and Features===
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<partinfo>BBa_K3866001 SequenceAndFeatures</partinfo>

Latest revision as of 19:31, 25 September 2021


pTrc:RBS GB compatible with A1-B2

The trc promoter is a hybrid of the lac and trp promoters which is a stronger promoter in comparison to the lac promoter.

Figure 1. The level 0 module : pupd2- pTrc:RBS (illustration from SnapGene)

Usage and Biology

The trc promoter was created by insertion of 1 bp into the 16 bp sequence between the consensus -35 and -10 sequences of the tac promoter, to obtain the optimal consensus distance of 17 bp between the -35 and -10 signal. This promoter includes the Lac Operator sequence lacO, which can be bound by the Lac repressor lacI, as well as an RBS BBa_B0034. For inducible gene expression from the trc promoter the lac repressor (LacI) is necessary. The repressor binds to the operator DNA with high specificity and inhibits gene expression until an inducer like allolactose or the analog IPTG (Isopropyl-?-D-thio-galactoside) is added to the medium. The inducer binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNA polymerase (RNAP) to transcribe the following genes and thereby leading to high levels of the encoded proteins.

Design Notes

The sequence was domesticated. We removed BsmBI, BsaI, BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for Golden Braid cloning. This has position A1-B2.

Figure 1.The overhangs of this part in the GoldenBraid Grammar

Verification of cloning

Figure 3. (C= Cut, U=Uncut) Restriction digestion of pTrc:RBS (C1 + C2) with: EcoRI + EcoRV, Expected bands: 1280bp, 896bp, Positive result: C1 + C2

Source

Synthesized by IDT.

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
    COMPATIBLE WITH RFC[1000]