Part:BBa_K1114702

BioBrick adaptation of MoClo Level 1 Reporter

This is a BioBrick adaptation of a Level 1 MoClo device that constitutively expresses RFP. The MoClo version of this device is BBa_K1114500.

This transcriptional unit contains the MoClo Level 0 parts J23104_AB, BCD1_BC, E1010m_CD, and B0015_DE within the pSB1C3 backbone. In the given sequence the internal fusion sites are incorrectly duplicated. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. See Level 0 pages for further information.

This device was converted into a BioBrick standard device using PCR to simultaneously remove the MoClo 5' and 3' sequences and insert the BioBrick 5' and 3' ends in their place. The PCR product was purified and digested with XbaI and PstI, and ligated into pSB1C3 (which was also cut with XbaI and PstI).

This part contains a bicistronic design (BCD) element as the 5' UTR regulatory part from Mutalik et al., 2013. From the library of BCDs, this devices contains a MoClo version of BCD1 (BioFAB # apFAB681) and we obtained the sequence data for this part from the BioFAB. The MoClo version of BCD1 is BBa_K1114106.

Characterization

We characterized this part using flow cytometry. The cells were grown overnight into stationary phase prior to a 1:200 dilution into 1XPBS, which was then run on a SORP LSRFortessa machine. We then used the TASBE tools to analyze our data and convert our readings into molecules of equivalent fluorescin (MEFL).

By converting the flow cytometry data into MEFLs, we now have an absolute unit of measurement for comparison.

The data is binned by fluorescence intensity and the tool also provides the cell count for each bin. We can see the population has a wide distribution of expression (top graph, blue bars).

The bottom graph shows the same data as the one above, but this is now ordered by cell count, from highest (dark purple) to lowest (pale purple).

We can now see that within the population of cells, the smallest binned group shows the highest level of expression. This may be due to higher copy counts of plasmids within that subpopulation of cells.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 11
  Illegal NheI site found at 34
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 694
  Illegal AgeI site found at 806
  
• 1000
  COMPATIBLE WITH RFC[1000]