Part:BBa_K3453104

sfGFP-LVAtag expression casette under the control of the T7 promoter

This part is an sfGFP-LVAtag (BBa_K2675006) expression cassette under the control of the T7 promoter BBa_K2150031.

Usage and Biology

In this composite part sfGFP-LVAtag (BBa_K2675006) was equipped by a custom made RBS (BBa_K2675017) designed using the Salis Lab RBS calculator v2.0 [1, 2] and placed under the control of the T7 promoter (BBa_K2150031) and of the strong SBa_000587 synthetic terminator (BBa_K3453000).

We used this part as a positive control of sfGFP-LVAtag expression.

The pictures of E. coli BL21 Star™(DE3) cells harbouring this part in pSB3T5 backbone show a very strong sfGFP-LVAtag expression (Figure 1) and the quantitative measurements confirm this (Figure 2). Moreover, sfGFP-LVAtag expression from this part is high and at a level comparable to that of the other positive control we used in our studies, the K3453105 in which the sfGFP-LVAtag was equipped by the synthetic stem-loop containing an RBS designed by Pardee et al. [3] to be part of a standard toehold switch (BBa_K3453005). This suggest that the two RBS (BBa_K2675017 and BBa_K3453005) are strong and have comparable strength.

In contrast, no fluorescence was detected in the negative controls as either the sfGFP gene was not present (empty pSB3T5 backbone) or the promoter and the RBS were absent (BBa K3453103).

Figure 1. Pictures of E. coli BL21 Star™(DE3) cells harbouring this part in the pSB3T5 backbone.

Figure 2. In vivo characterisation of sfGFP expression by E. coli BL21 Star™(DE3) cells harbouring the positive controls of sfGFP-LVAtag expression BBa K3453104 and BBa K3453105. The negative controls have been performed with an empty pSB3T5 and BBa K3453103 (no promoter, no RBS). Fluorescence values were normalised by OD_600nm and, using the calibration curves presented on the Measurement page of our wiki, the arbitrary units were converted into Molecules of Equivalent FLuorescein (MEFL) / particle. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

References

[1] Espah Borujeni A, Channarasappa AS, Salis HM. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res (2014) 42, 2646-2659.

[2] Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol (2009) 27, 946-50.

[3] Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, Daringer NM, Bosch I, Dudley DM, O'Connor DH, Gehrke L, Collins JJ. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell (2016) 165, 1255-1266.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 75
  
• 1000
  COMPATIBLE WITH RFC[1000]