Difference between revisions of "Part:BBa K2319009"

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Revision as of 14:34, 17 October 2018

6xHis-mCherry under T7 expression system

This part expresses 6xHis-mCherry using our T7 expression system.

Usage and Biology

Using this as a T7 expression backbone

Expressing any protein of interest

This T7 expression backbone can be used to express any protein if its coding sequence (with a start codon) is inserted using the HindIII and NheI sites. These sites can be added to the coding sequence using PCR with primers having 5'-overhangs.

Fusing a protein domain at the N-terminus of an existing protein

By inserting the coding sequence of a protein domain (including the start codon) using the HindIII and AgeI sites into the T7 expression backbone (which already contains a protein coding sequence), an N-terminal fusion can be performed.

Expressing a protein of interest

No system is better for protein expression than E. coli BL21 (DE3) as its lon and ompT protease deficiency yields a huge amount of protein. BL21 (DE3) is a lysogenic strain that has the T7 RNA polymerase gene integrated into its genome under the lac operon; adding IPTG induces expression of T7 RNA polymerase, which recognizes the T7 promoter sequence. Any gene inserted downstream of the T7 promoter can thus be expressed.

Transform this part into BL21 (DE3) and induce protein overexpression with IPTG after the culture reaches early-log to mid-log phase (optimize this by inducing at different times).

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 crdue 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 intesity (rough esimation):

	% 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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Sequence and Features