Part:BBa_K137040

Coding SSM device: GFP with (AGTC)10 after start codon

Strong promoter + strong RBS + GFP with (AGTC)10 repeat after start codon + double terminator

Usage and Biology

As part of the Caltech 2008 iGEM [http://2008.igem.org/Team:Caltech/Project/Population_Variation population variation project], Parts K137040 to K137043 were placed on pSB1A2 and transformed into DH10B. When measured on a plate reader (Tecan Sapphire), they gave the following fluorescence values:

When measured on a flow cytometer using the Caltech iGEM [http://2008.igem.org/Team:Caltech/Protocols/Flow_cytometry flow cytometry protocol], they produced the following distributions:

• 

  The fluorescence spectrum of individual cells containing the AGTC ‘in’ and ‘out’ coding SSM constructs. Spectrum of positive control is in green, negative control is in blue, AGTC ‘in’ construct is in brown, and AGTC ‘out’ is in red. Note that the ‘in’ repeat spectrum has a peak at a higher value than the ‘out’ repeat spectrum. In addition, note the broad distribution of the spectrum. The constructs were placed in a high copy plasmid (>100 copies per cell). Thus, each one of those plasmids has a chance of slipping, and the total number of GFP transcripts from all the plasmids in a cell determines the overall fluorescence of the cell.

• 

  The fluorescence spectrum of individual cells containing the TA ‘on’ and ‘off’ regulatory SSM constructs. Spectrum of negative control is in red, TA ‘on’ is in blue, and TA ‘off’ is in green. The TA ‘on’ distribution lies to the right of the TA ‘off’ distribution, indicating that the TA ‘on’ construct is a better promoter than its counterpart. These distributions were taken at a higher PMT value than the distributions in the left image. The peaks in between 10⁰ and 10¹ fluorescence may be debris the flow cytometer picked up. Our flow cytometer is commonly used with eukaryotic cells and has not yet been optimized for use with bacterial cells. The cells measured with the flow cytometer in this figure are the same ones measured with the plate reader.

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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 745