Difference between revisions of "Part:BBa J18932"

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	<partinfo>BBa_J18932 parameters</partinfo>		<partinfo>BBa_J18932 parameters</partinfo>
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		+
		+	==Contribution: UniGE-Geneva==
		+	'''Group:''' UniGE-Geneva
		+	<br>'''Authors:''' Grégory Segala and Bryan Bourrat
		+	<br>'''Summary:''' : In the list of Parts assigned to our team, we decided to characterize the fluorescent protein : mCherry RFP (BBa-J18932). Our goal was to observe the evolution of the intensity of fluorescence emitted by this protein, after induction by IPTG (Isopropyl -D-1-thiogalactopyranoside), varying two parameters: temperature and pH.
		+	<br>'''Documentation:'''
		+	<br>
		+
		+	[[Image:T--UniGE-Geneva--unige-igem-characterization-2.004.png|frame|none|'''Fig 1''':Procedure details of the experiment.]]
		+	[[Image:T--UniGE-Geneva--unige-igem-characterization-2.005.png|frame|none|'''Fig 2''':Variation of Fluorescence Intensity depending on Temperature.]]
		+	[[Image:T--UniGE-Geneva--unige-igem-characterization-2.006.png |frame|none|'''Fig 3'''::Variation of Fluorescence Intensity depending on pH.]]

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Revision as of 13:23, 21 October 2019

mCherry RFP

Red fluorescent protein derived from DsRed.

Advantages:

• fast folding and maturation
• bright and photo-stable

Purity issues (update):

• Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli
• Contains a hidden translation start site in the N-terminal -> up to 50% of protein produced in E.coli will be truncated (R. Grünberg, unpublished)
• SDS treatment and boiling before PAGE hydrolyzes the chromophore at F//MYG splitting the protein in half (discussed in Gross et al. (2000) The structure of the chromophore within DsRed protein from coral.)
• Best maturation for expression at 20 or 25 C (rather than 37)

Usage and Biology

Characterization

Expression with BBa_K2319009

The protein was expressed under T7 promoter in E.coli BL21(DE3) with 6x-His tag at the N-terminal. The culture was induced at 37°C for three hours with a final IPTG concentration of 500μM. The cells were then lysed to obtain the protein. The size of the complete protein with 6x-Histag is about 26kDa. We observed two bands in the induced sample between 25 kDa and 32 kDa. The heavier band is the non-truncated protein and the lighter one is its truncated counterpart.

Purification using Ni-NTA with BBa_K2319009

The cell lysate thus obtained was purified using Ni-NTA beads which only bind to proteins with a 6x-His tag, which is absent in the truncated protein. Ideally, the supernatant after binding should have the truncated protein and the elution after purification should have the non-truncated protein. This however is not true because the binding of 6xHis to Ni-NTA is not perfect.

Fluoroscence

Excitation Spectrum

The excitation spectrum of the purified sample (elution) was obtained at a fixed emission wavelength of 610 nm. The excitation maxima was obtained at 567 nm.

Emission Spectrum

The emission spectrum of the purified sample (elution) was obtained at a fixed excitation wavelength of 587 nm. The emission maxima was obtained at 605 nm

Quantification of Truncation

The truncation of mCherry was determined by through two different methods:

• By analysing the intensity of the truncated and non-truncated protein bands from the SDS PAGE of the crude lysate.(rough estimation)
• By combining the fluorescence and gel intensity data of the Ni-NTA purification fractions (supernatant after binding, wash and elution).This is done assuming that truncated and non-truncated protein has the same fluorescence. The fluorescence of each of the above fractions was divided into fluorescence due to truncated and non-truncated protein based on their corresponding band intensities. The sum of fluorescence values of truncated and non-truncated protein were then used as a measure of their concentration to determine truncation.

Truncation Data

From gel intensity (rough estimation):

	% of protein
	Truncated	Non-Truncated
Replicate 1	30.25	69.75
Replicate 2	48.6	51.4
Replicate 3	35.8	64.2
Replicate 4	34.2	65.8
Replicate 5	49.8	50.2
Average	39.73	60.27
Std.dev	7.95	7.95

From this, mcherry is estimated to have a truncation of 39.73 % ± 7.95 %

From the calculations combining gel intensity and fluorescence:

	% of protein
	Truncated	Non-Truncated
Replicate 1	34.66	65.34
Replicate 2	38.91	61.09
Replicate 3	42.53	57.47
Replicate 4	39.51	60.49
Replicate 5	38.47	61.53
Average	38.82	61.18
Std.dev	2.52	2.53

From this, mcherry is estimated to have a truncation of 38.82 % ± 2.52 %

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Improvement by IISc-Bangalore 2018

The sequence was modified to reduce the strength of the internal RBS and made into BBa_K2609006.

Improvement by IISc-Bangalore 2019

The sequence was modified to reduce the truncation by replacement of the internal start codon with Leucine or Isoleucine and made into BBa_K3165012 and BBa_K3165013 respectively.

Sequence and Features

Contribution: UniGE-Geneva

Group: UniGE-Geneva
Authors: Grégory Segala and Bryan Bourrat
Summary: : In the list of Parts assigned to our team, we decided to characterize the fluorescent protein : mCherry RFP (BBa-J18932). Our goal was to observe the evolution of the intensity of fluorescence emitted by this protein, after induction by IPTG (Isopropyl -D-1-thiogalactopyranoside), varying two parameters: temperature and pH.
Documentation: