Difference between revisions of "Part:BBa K4345003"

Revision as of 13:02, 12 October 2023

mPapaya

mPapaya is a constitutively fluorescent protein with an excitation wavelength of 530 nm and an emission wavelength of 541 nm.

Usage and Biology

Whilst waiting for the parts designed for our project to be synthesised, we wanted to get into the lab and familiarise ourselves with the equipment and techniques. Our supervisor suggested we practised transforming, growing and expressing proteins in bacteria with fluorescent proteins. These are relatively non-toxic to bacteria and easy to visualise which allows for conformation of transformation and protein expression. During training on the imaging flow cytometer, we noticed some unusual results. These are presented below and are our bronze medal contribution to the iGEM registry.

Expression of sfGFP (Pedelacq et al., 2006), mCherry (Shaner et al., 2004) and mPapaya1 (Hoi et al., 2013) was under control of the strong constitutive promoter BBa_J23100, combined with the strong RBS BBa_B0034 and terminated by the double terminator BBa_B0015. The gene was carried on a medium copy number plasmid with ampicillin resistance and transformed into E. coli DH5𝛼. 5 mL /50 mL (delete as appropriate) cultures were grown at 37 C, 200 rpm for 18 h. Cell morphology was investigated using an Image Stream Mark II (Amnis-Luminex Corp.) Imaging Flow Cytometer configured with Bright Field (white light), Side-Scatter (785 nm) and either GFP (excitation laser 488 nm, emission 533/55 nm), RFP (excitation laser 592 nm, emission 610/30 nm) or YFP (excitation laser 488 nm, emission 702/85 nm).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 267
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 267
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 267
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 267
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 267
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

mPapaya is derived from Zoanthus sp. The excitation and emission spectra are presented below.

This picture was obtained from fpbase.org.

References

mPapaya. (2022). FPBase. Retrieved July 7, 2022, from https://www.fpbase.org/protein/mpapaya/

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4345003 short</partinfo>
-mPapaya is a constitutively fluorescent protein with an exctitation wavelength of 530 nm and an emission wavelength of 541 nm.
+mPapaya is a constitutively fluorescent protein with an excitation wavelength of 530 nm and an emission wavelength of 541 nm.
+===Usage and Biology===
+Whilst waiting for the parts designed for our project to be synthesised, we wanted to get into the lab and familiarise ourselves with the equipment and techniques. Our supervisor suggested we practised transforming, growing and expressing proteins in bacteria with fluorescent proteins. These are relatively non-toxic to bacteria and easy to visualise which allows for conformation of transformation and protein expression. During training on the imaging flow cytometer, we noticed some unusual results. These are presented below and are our bronze medal contribution to the iGEM registry.
+Expression of sfGFP (Pedelacq et al., 2006), mCherry (Shaner et al., 2004) and mPapaya1 (Hoi et al., 2013) was under control of the strong constitutive promoter [https://parts.igem.org/Part:BBa_J23100 BBa_J23100], combined with the strong RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] and terminated by the double terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. The gene was carried on a medium copy number plasmid with ampicillin resistance and transformed into <i>E. coli</i> DH5𝛼. 5 mL /50 mL (delete as appropriate) cultures were grown at 37 C, 200 rpm for 18 h. Cell morphology was investigated using an Image Stream Mark II (Amnis-Luminex Corp.)  Imaging Flow Cytometer configured with Bright Field (white light), Side-Scatter (785 nm) and either GFP (excitation laser 488 nm, emission 533/55 nm), RFP (excitation laser 592 nm, emission 610/30 nm) or YFP (excitation laser 488 nm, emission 702/85 nm).
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