Part:BBa_K2348002

mCardinal

mCardinal obtained from Addgene [1]
This part was used in our acide induced construct K2348011. In this construct mCardinal is expressed but we were unable to detect any flourescence in E. coli. This might be because, as we later noticed, mCardinal is designed for mammalian expression.

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 BsaI.rc site found at 445
  Illegal BsaI.rc site found at 634
  

Cornell 2020 Literature Research and Modeling

Note: All work on this basic part was done virtually through literature research due to COVID-19 restrictions.

mCardinal is a red fluorescent protein that absorbs light of peak wavelength 604 nm and emits light of peak wavelength 659 nm, derived from Entacmaea quadricolor. This BioBrick part consists of the mCardinal coding gene [1] with stop codon TAA. The sequence was reverse translated with codon optimization for E. Coli. It is driven by a constitutive promoter (BBa_J23100), has a ribosome binding site (BBa_J61100), and a terminator (BBa_B0015). The constitutive promoter was chosen to allow for maximum and constant mCardinal expression. After reviewing several sources, we were able to find that mCardinal could realistically be produced by E. coli cells. Chu et al (2014) was able to study the mCardinal fluorescent protein after purifying it from cultured E. coli cells that were grown to produce the protein [2].

In order to determine the location of the E. coli in the body, our system utilized a fluorescent reporter that can be detected by our sensing device, Trichoscan. We chose to use a red fluorescent protein due to the ease of detection; red light can best penetrate layers of the skin [3], and mCardinal is a relatively bright red fluorescent protein. Thus, the Trichoscan device can determine the location of the bacteria.

Figure 1. mCardinal protein production process under a constitutive promoter. The mCardinal gene produces a fluorescent protein that absorbs light at a peak wavelength of 604 nm and emits light at a peak wavelength of 659 nm. Our models account for the translation rate and degradation rate of the mRNA in order to determine a steady state concentration of the mCardinal protein.

Under the assumption that mCardinal can be expressed in prokaryotes as we noted in literature, we modeled the output of the mCardinal protein using the following differential equation, and we solved it using MATLAB’s ode45 function. The equation represents the production of mCardinal protein. The first term represents the basal expression under the constitutive promoter (BBa_J23100), while the second represents protein degradation.

The MATLAB scripts corresponding to the mCardinal modeling can be downloaded here:

File:ModelingCodeCornelliGEM2020.zip

Variable	Parameter	Value	Source
[mCardinal]	Concentration of mCardinal	--	--
basT	Basal transcription rate	23.22 micromolar/minute	[4]
dC	degradation rate of mCardinal	0.575 min-1	[5]

The modeling of the mCardinal protein output shown in Figure 2 indicates that the protein reaches a steady state concentration of approximately 403 μM. At this concentration, Trichoscan can detect the bacteria at a depth of around 20mm.

Figure 2. This plot depicts the concentration of mCardinal protein produced over time. mCardinal is produced continuously by E. coli; the concentration of mCardinal rises to a steady-state concentration of about 403 μM before its degradation rate matches its production rate. At this concentration, the protein’s fluorescence can be detected externally by Trichoscan.

References:

[1] PcDNA3-mCardinal Sequences (2). (n.d.). Retrieved October 17, 2020, from https://www.addgene.org/51311/sequences/

[2] Chu, J., Haynes, R. D., Corbel, S. Y., Li, P., González-González, E., Burg, J. S., . . . Lin, M. Z. (2014). Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nature Methods, 11(5), 572-578. doi:10.1038/nmeth.2888

[3] Ash, C., Dubec, M., Donne, K., & Bashford, T. (2017). Effect of wavelength and beam width on penetration in light-tissue interaction using computational methods. Lasers in medical science, 32(8), 1909-1918.

[4] Yildirim, N., & Mackey, M. C. (2003). Feedback Regulation in the Lactose Operon: A Mathematical Modeling Study and Comparison with Experimental Data. Biophysical Journal, 84(5), 2841-2851. doi:10.1016/s0006-3495(03)70013-7

[5] Lambert, T. (n.d.). MCardinal at FPbase. Retrieved October 14, 2020, from https://www.fpbase.org/protein/mcardinal/