Part:BBa_K4907147

J23100-B0034-rfp

Biology

This part encodes a monomeric far-red fluorescent protein derived from Entacmaea quadricolor, which has excitation and emission wavelengths at 588 and 633 nm respectively. It is a commonly used reporter for expressing and tracing (1).

Usage and design

Our co-culture system uses this part as a growth state indicator for E. coli Nissle 1917. We opted to use pET-28a(+) as the vector of rfp and then transformed it into the bacteria.

Fig. 1 Gene circuit of BBa_K4907147

This part also served as a reporter of the characterization for a range of VSW-3 RNAP expression systems BBa_K4907108, BBa_K4907110, BBa_K4907111, BBa_K4907113 as well as our "AND GATE" based on the VSW-3 RNAP.
Please click the composite part directly for more detailed information.

Characterization

Agarose gel electrophoresis (AGE)

The promotor (BBa_J23100) and the RBS (BBa_B0034) were ligated with this part and then constructed into a pET-28a(+) vector through seamless cloning. Colony PCR and gene sequencing were used to verify that the transformants were correct when constructing this circuit, and target bands (849 bp) can be observed between 1000bp and 750bp (Fig. 2). The correct plasmid was transformed into E. coli Nissle 1917 for further characterization.

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907037_pET-28a(+)

The growth curve of RFP in EcN

We measured the OD₆₀₀ and the fluorescence intensity of different EcN carrying J23100-B0034-rfp-T7t_pET-28a(+). These three bacteria were grown overnight in Lysogeny Broth (LB) containing kanamycin (100 µg/mL) at 37 °C and 200 rpm. Cultures were inoculated in fresh LB at 2% (v/v). Samples were always made in triplicates and a blank of LB. During 8h the absorbance at OD₆₀₀ and fluorescence (excitation 588 nm and emission 633 nm) were measured with intervals of 1 hour.

Fig. 3 OD₆₀₀-t graph of different types of EcN. Fig. 4 The curve of fluorescence intensity in different EcN

We can see from the graphs above, that the fluorescence intensity has a similar trend with OD₆₀₀, which means the fluorescence intensity can reflect the strain density under certain conditions. E. coli BL21(DE3), another bacterium in our co-culture system, in which we introduced gfp (BBa_K4907146) gene to produce green fluorescence, a similar trend was also observed. That met our expectations, in this case, it’s possible for us to control the ratio of the two co-cultured bacteria by controlling the fluorescence of each.
Therefore, we fitted the linear regression curves of FI and OD₆₀₀ (Fig. 5) to be able to convert the proportion of red and green fluorescence values we tested to the ratio of both bacteria in the co-culture system.

Fig. 5 Linear curves of Red fluorescence intensity and the OD₆₀₀ (R²=0.9289)

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Reference

1. D. Shcherbo et al., Far-red fluorescent tags for protein imaging in living tissues. Biochem J 418, 567-574 (2009).

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 488
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 610
  
• 1000
  COMPATIBLE WITH RFC[1000]