Difference between revisions of "Part:BBa K3866022"

 
(→‎Design Notes)
 
(5 intermediate revisions by the same user not shown)
Line 5: Line 5:
 
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.
  
−
<!-- Add more about the biology of this part here
+
[[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===
  
−
<!-- -->
+
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.
−
<span class='h3bb'>Sequence and Features</span>
+
−
<partinfo>BBa_K3866022 SequenceAndFeatures</partinfo>
+
  
 +
===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 <bbpart>BBa_K3505007</bbpart> and has overhangs compatible for Golden Braid cloning.
 +
This has position A1-B2.
  
−
<!-- Uncomment this to enable Functional Parameter display
+
[[File:T--Thessaly--GB-GGAG-AATG.jpeg|800px|thumb|none|<i><b>Figure 2.</b> The overhangs of this part in the GoldenBraid Grammar</i>]]
−
===Functional Parameters===
+
 
−
<partinfo>BBa_K3866022 parameters</partinfo>
+
===Verification of cloning===
−
<!-- -->
+
 
 +
[[File:T--Thessaly--pTrc pLac--digestion.png|500px|thumb|none|<i><b>Figure 3.</b> (C= Cut, U=Uncut) Restriction digestion of pLac:RBS with: EcoRI + EcoRV, Expected bands: 1279bp, 896bp, Positive result: C3 + C4</i>]]
 +
 
 +
===Source===
 +
Synthesized by IDT.
 +
 
 +
===Sequence and Features===
 +
<partinfo>BBa_K3866022 SequenceAndFeatures</partinfo>

Latest revision as of 19:56, 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 2. The overhangs of this part in the GoldenBraid Grammar

Verification of cloning

Figure 3. (C= Cut, U=Uncut) Restriction digestion of pLac:RBS with: EcoRI + EcoRV, Expected bands: 1279bp, 896bp, Positive result: C3 + C4

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]