Difference between revisions of "Part:BBa K3866022"
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This lac promoter includes a CAP binding site and the lac operator sequence lacO.
 
This lac promoter includes a CAP binding site and the lac operator sequence lacO.
  
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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>]]
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[[Image:T--Thessaly--placsnap.png|800px|thumb|none|<I><b>Figure 1.</b> The level 0 module : pupd2- pLac: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.
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The lac promoter is not as strong as the tac or the trc promoter, but in high copy-number vectors it allows expression of foreign proteins at respectable levels. In the absence of the lac repressor (LacI) expression from the lac promoter is turned on. Maximum induction of the lac promoter requires the action of the cAMP activator protein (CAP, crp gene product), which is most active when cells are grown in medium lacking glucose. Media that contain glucose as carbon source should not be used to express genes from this promoter. For inducible gene expression from the lac 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===
 
===Design Notes===
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===Verification of cloning===
 
===Verification of cloning===
  
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[[File:T--Thessaly--pTrcRBS--digestion.png|300px|thumb|none|<i><b>Figure 3.</b> (C= Cut, U=Uncut) Restriction digestion of pTrc:RBS with: EcoRI + EcoRV, Expected bands: 1280bp, 896bp, Positive result: C1 + C2</i>]]
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[[File:T--Thessaly--pLacRBS--digestion.png|300px|thumb|none|<i><b>Figure 3.</b> (C= Cut, U=Uncut) Restriction digestion of pTrc:RBS with: EcoRI + EcoRV, Expected bands: 1280bp, 896bp, Positive result: C1 + C2</i>]]
  
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===
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===Sequence and Features===
 
===Sequence and Features===
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<partinfo>BBa_K3866001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3866022 SequenceAndFeatures</partinfo>

Revision as of 19:21, 25 September 2021


pLac:RBS GB compatible with A1-B2

This lac promoter includes a CAP binding site and the lac operator sequence lacO.

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

Usage and Biology

The lac promoter is not as strong as the tac or the trc promoter, but in high copy-number vectors it allows expression of foreign proteins at respectable levels. In the absence of the lac repressor (LacI) expression from the lac promoter is turned on. Maximum induction of the lac promoter requires the action of the cAMP activator protein (CAP, crp gene product), which is most active when cells are grown in medium lacking glucose. Media that contain glucose as carbon source should not be used to express genes from this promoter. For inducible gene expression from the lac 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

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

Experimental Use and Experience

This part is used in BBa_

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]