Part:BBa_K2002001

Infared Fluorescent Protein

iRFP is an infrared fluorescent protein that requires an excitation wavelength of approx. peak 660 nm and an emission wavelength of approx. 710 nm. The iRFP protein was used in the experiment which is slightly red-shifted when compared with iRFP 1.4. (Filonov et al.)

iRFP requires the presence of a co-factor billiverdin to fluoresce. Billiverdin is formed through the breakdown of haeme and is present in the cytosol of mammalian cells. Billiverdin is normally converted to bilirubin via biliverdin reductase and lipophilic reactive species catalyse the reverse reaction.

IRFP construct

The plasmid we used in our experiments had to be capable of replicating in L.lactis. We also needed to ensure that there was suitable antibiotic resistance. This plasmid contains the P44 promoter which is a strong promoter in L.lactis. It also has chloramphenicol resistance gene, RepA and RepC replication initiation genes. We used Gibson cloning to ligate in the IRFP construct into a XbaI site which is downstream of the P44 promoter.

Materials & Methods:

RAW 264.7, a mouse macrophage cell line, were used in this experiment. Co-incubation of the L.lactis strain with the macrophages led to phagocytosis of the bacteria. An Odyssey scanner can be used to image the fluorescent read out. Our procedure consisted of - seeding the macrophages at a density of 1 x 105 cells per well in a 12 well plate. The total amount of culture medium in each well was 1 ml. The RAW 264.7 cells were allowed to grow for four hours before bacteria was added to the wells. - L.lactis was subcultured from a fresh overnight culture and grown to an OD of 1. An OD to CFU curve was created for this strain of L.lactis in order to convert the OD 600 reading into the number of colony forming units. - Three different multiplicity of infections were used in the experiments (100:1, 500:1, 1000:1 in the initial experiments and 100:1, 300:1, 1000:1 in the later experiments). Following addition, the RAW 264.7 cells and L.lactis were left at 37 degrees Celsius 5 % CO2 for 4 hours. - After this co-incubation penicillin/ streptomycin was added to each of the wells to kill the extracellular and intracellular bacteria and the plate was incubated for a further 1-2 hours. - A PBS wash was carried out and the plate was imaged in the Odyssey scanner.

Results

Delivery of iRFP protein to RAW 264.7 macrophage cells

Note in this experiment. There is no FBS in the media which if present can allow protein expressed by bacteria and not in mammalian cells to fluoresce. The above experiment was done to show that this protein is fluorescent in mammalian cells and it illustrated that the 300:1 MOI gave the highest IRFP signal, this would suggest that the higher number of L.lactis is inducing a cell stress that results in apoptosis. Cell viability studies would be necessary to determine the extent of the cell death and compare the amount of viable cells between the two conditions. However, this experiment suggests that the protein is inside the cytosol of the macrophage cells since there is fluorescence. The only source of the co-factor is now the cytosol of the macrophage cells.

Media supplementation: Adding FBS to media to allow for bacterial expressed protein to fluoresce

Since FBS contains the required co-factor, if it is added to media it can cause bacteria expressing iRFP to fluoresce. L.lactis were incubated in media + FBS for 4 hours and then analysed using the Odyssey scanner.

A graph of fluorescent intensity versus cell number was created to show that as the number of cells increase, the fluorescent intensity also increases proportionally.

References

Filonov, G. S., Piatkevich, K. D., Ting, L.-M., Zhang, J., Kim, K. and Verkhusha, V. V. 'Bright and stable near-infrared fluorescent protein for in vivo imaging'

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 227
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 757