Part:BBa_K4907146

J23100-B0034-gfp-B0015

Biology

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence. Its excitation wavelength is 488 nm, and its emission wavelength is 533 nm. When gfp is expressed in bacteria, it produces non-exocytosed GFP proteins that cause the bacteria to fluoresce green at an excitation wavelength of 488 nm. It is a commonly used reporter for expressing and tracing.

Usage and Design

As we intended to carried out co-culture fermentation of two engineered bacteria E. coli Nissle 1917 (EcNP type) and BL21(DE3) which produce BC and HA respectively, we realized that the ratio between BC and HA would directly affect the water holding capacity of the final product. Hence, after reviewing an article (1), we decided to regulate the strain ratio of two engineered bacteria by introducing fluorescent proteins to reflect the strain density under certain condition. (see more details on Designpage)
For E. coli BL21(DE3), we decided to introduce GFP into it. and hence BBa_J23100, BBa_B0034, BBa_K4907036 and BBa_B0015 were used to construct corresponding circuit BBa_K4907146, which was assembled on pSB3C5 by standard assembly (Fig. 1). After successful construction and gene sequencing, we transformed the plasmid into E. coli BL21(DE3) for further verification and measurement.

Fig. 1 Graphic descirption of genetic circuit BBa_K4907146

Characterization

Agarose Gel Electrophoresis

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (1195 bp) can be observed at the position between 2000 and 1000 bp.

Fig. 2 DNA gel electrophoresis of the colony PCR products.BBa_K4907146_pSB3C5 in E. coli BL21(DE3). Target bands (1195 bp) can be observed at the position between 1500 bp and 1000 bp.

Fluorescence intensity versus time measurement

The bacterial culture was cultivated overnight, then the measurement of OD₆₀₀ and fluorescence intensity was carried out in triplicate.

Fig. 3 a Growth curve of E. coli BL21(DE3) with BBa_K4907146. b Fluorescent intensity curve of E. coli BL21(DE3) with BBa_K4907146.

The graph showed us that the fluorescence intensity has a similar trend with OD₆₀₀ through out the experiment, which means the fluorescence intensity can reflect the strain density under certain condition.

Reference

1. J. Gutiérrez Mena, S. Kumar, M. Khammash, Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback. Nat. Commun. 13, 4808 (2022).

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
  COMPATIBLE WITH RFC[1000]