Part:BBa_K5299200

P3.1 - BBa_B0030 - BBa_I746916 - BBa_B0015

Description

It consists of a promoter[1], a RBS, a sfGFP and a terminator. The P3.1 BBa_K4583008 is a promoter autoinducible in the stationary phase, as described in the work of Jaishankar et al. (2020).[1]

Usage and Biology

It is able to produce the sfGFP according to the promoter's abilities.

It was constructed in order to check the strength of the P3.1 promoter. In the future, it can be used by other teams that wish to utilise the P3.1 promoter, in order to estimate that P3.1 is a suitable candidate in their design for their chassis of choice.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 259
  

Results

Measurements for OD 600nm and fluorescence (488nm,510nm[2]) were taken over the course of 16 hours, in a 96-well microplate (black plate, clear bottoms). Clonings were done according to the Golden Braid method, leaving us with level 1 constructs in the pDGB3a1 backbone BBa_K4213058.

Construct transformed into E.coli BL21 (DE3) chassis, incubated at 37oC, 180rpm for 16 hours.

Plated 120 ul 5 times, out of each single liquid bacterial culture, created from the same bacterial colony, in order to establish accuracy through technical repeats.

Medium used was M9 due to minimal interference, with D-glucose serving as the carbon source. Also, served as blank, plated 5 times.

Measurements were normalised as such: using the average price of fluorescence for the 5 technical repeats and dividing it by the average price of OD.
Standard deviation included in the graphs.

Measurements were taken over the course of 16 hours. Timepoint 0 is depicted as 1h, Timepoint 1 is depicted as 2h, etc. Here are the results for this part.

Figure 1: Constructs with the BBa_J23119 or the BBa_K4583008 or the BBa_J45992 promoter, the BBa_B0030 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. pDGB3a1 serves as a negative control. BBa_J23119 and BBa_J45992 serve as phase activation controls. BBa_J45992 also serves as strength control, due to the same phase activation.

References

• [1]: Jaishankar, J., & Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 109 (102491), 102491. doi:10.1016/j.plasmid.2020.102491
• [2]: Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24 (1), 79–88. doi:10.1038/nbt1172