Part:BBa_K1030000

J23100->tetA-asv+sfGFP

We use a strong synthetic constitutive promoter, J23100 to drive the expression of tetA, an tetracycline transporter that can also transport toxic nickel ion into cell which reduce e coli growth rate and kill cells. an asv degradation tag is added to the c-terminal of tetA. A superfolder GFP is expressed under the control of the same promoter so that we can use fluorescent plate reader, flow cytometer and microscope to monitor the expression level of tetA. They are not fused.

We generated plasmids, in which tetA and GFP are under control of 8 constitutive promoters of various intensities. There are J23100 through J23107.

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We first check the growth curve of E coli containing our biobricks with various tetracyclin concentration. We grow then in Luria Broth (LB) using plate reader. The data is shown in biobricks section. With gradually increasing in tetracylcin concentration,the growth is slowing down and eventually turning negative. We fit the growth rate to the exponential growth phase of each sample and plot them against the mean GFP level, which representing the tetA level. It clearly shows the positive correlation between growth rates of E coli and the levels of tetA, an transporter than pump tetracyclin out of E coli.

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We then added Nickel Chloride of various concentrations to the E coli containing the biobricks above, and growth them in Luria Broth (LB) using fluorescent plate reader. The growth curves are shown in the biobricks section. With increasing in Nickel concentration, the growth is also slowing done to negative. We then plot the growth rate against the mean GFP level. Most of the biobricks fit the negative relation between tetA and growth rate with Nickel. However, the trend is not as robust as with tet. In addition, even with lowest tetA level, there is still toxic effect with nickel, which reduced the significance of tuning growth rate via tuning tetA. We will try to find out why.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1106
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 960
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 406
  Illegal NgoMIV site found at 566
  Illegal NgoMIV site found at 934
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1273