Difference between revisions of "Part:BBa K731500"
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===Usage and Biology===
 
===Usage and Biology===
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LacI gene product is a trascriptional inhibitor inactivated by allolactose, a lactose metabolite. In its active conformation (no allolactose bound) it recognize and bind ''lac'' operator sterically preventing RNA polymerase binding to the ''tac'' promoter and owering constitutive gene expression. In its active conformation (allolactose bound) it releases ''lac'' operon, allowing polymerase recognition of P''tac''.
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In our experiences we have used isopropyl β-D-1-thiogalactopyranoside (IPTG, an allolactose analogous) instead of lactose to induce protein expression.
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This part allows IPTG-induced gene expression. ''lac'' operon and P''tac'' activity has been measured by placing a GFPmut3b under control of this regolative part and assaying protein level by fluorescence. See [https://parts.igem.org/Part:BBa_K731520 BBa_K731520] for sequence details.
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This part has been characterize in both pSB4K5 and pSB1C3.
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<div style="text-align:center;">[[Image:]]</div><br/>
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'''FIGURE 1. Protein expression and cell growth upon IPTG induction''' NEB10b cells transformed with BBa_K731520 were grown in LB at 37°C until OD of 0.6 and induced with different concentrations of IPTG. After 4 hours a 1.5 mL aliquot was taken from each sample. Cells were span down and resuspended in 1.5 mL of PBS. Panel A: Optical density at 600nm and intensity of fluorescence in pSB1C3. Panel B: Optical density at 600nm and intensity of fluorescence in pSB4K5.
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Fluorescence measurements were taken with a Varian Cary Eclipse Spectrophometer using an excitation wavelenght of 464 nm
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<div style="text-align:center;">[[Image:AT1480_2.jpg]]</div><br/>
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'''FIGURE 2. Effects of carbon source and induction on protein expression levels.''' Cells were grown at 37°C in LB until it was reached an OD of 0.4. The cells were at this point span down and resuspended in an equal volume of MOPS medium and allowed to grow to an OD of 0.6. Prior induction the cells were splitted into two samples of equal volume and one of the two sample was induced with 0.1 mM IPTG. Every hour a 1.5 mL aliquot was taken to measure optical density and fluorescence intensity. The assay was performed in two different MOPS media. MOPS A: 60 mM glycerol ("hot" colours). MOPS B: 30 mM glucose ("cold" colors).  The experiment has been performed in triplicate. Panel A: Optical density at 600nm and intensity of fluorescence in pSB1C3. Panel B: Optical density at 600nm and intensity of fluorescence in pSB4K5.
  
 
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Revision as of 12:37, 17 September 2012

[LacI + LacIq promoter] reverse + [tac promoter + lac operator] forward

This composite part consist of BBa_K731300 part (lacI-lacIq) and a tac promoter followed by a lac operator in the forward direction. It is a ready to use composite part and it allows the expression of a gene of interest with a strong E.coli promoter and under the control of IPTG. The lac operator decreases basal levels of expression.

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]