Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/labeling"

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Labeling

Short summary

For the labeling of a target protein in vivo a fluorescent amino acid should be incorporated through the amber stop codon. Our aim is to provide a tRNA/aminacyl-synthetase (tRNA/aaRS) to the iGEM community to easily incorporate the fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) ethylglycin (CouAA) during translation in response to the amber stop codon.
To demonstrate the advantages of this tool, we want to colocalize the ribulose 1,5‑bisphosphat carboxylase oxygenase (RuBisCo) from Halobacillus neaplitanus and the carboxysome in E. coli cells. The carboxysome is labeled with the green fluorescent protein (GFP) and the RuBisCo should be labeled with the fluorescent amino acid at different positions and with the red fluorescent protein (RFP) to compare the advantages and disadvantages of both labeling strategies.

Characterisation of the fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) ethylglycin (CouAA)

To find out if L‑(7‑hydroxycoumarin‑4‑yl) ethylglycin (CouAA) is suitable for cultivation and expression experiments we performed experiments on the stability and fluorescence ability. At first the absorbance of LB media containing 1 mM was measured at the beginning and after 24 hours incubation at 37 °C. It turns out that the absorbance maximum is at 328 nm and the loss of fluorescence is under 10 %, which can also be a result of the photobleaching of CouAA. After that we recorded a fluorescence spectra of the sample with constant irradiation at 328 nm. The fluorescence maximum was measured at 452 nm, both spectra are shown in figure 1. The absorbance and fluorescence spectra are very similar to the spectra published by Wang 2006. The small variances in the absorbance are probably caused by different absorptions of the used media. After incorporation in the protein we recorded the absorbance and fluorescence spectra of CouAA in the protein to, the spectra are shown in figure 3.

Figure 1: Absorbance and fluorescence spectra of CouAA, recorded by UV/VIS-sprectralphotometer. The fluorescece spectra was recorded at the absorbance maximum at 328 nm.

Evolved Tyrosine tRNA/aminoacyl-synthetase (TyrRS) for the incorporation of 7-hydroxy-L-coumaryl-ethylglycin (CouAA)

For the construction of the synthetase, we decided to use the Tyrosine tRNA/aminoacyl-synthetase from Methanococcus jannaschii with mutations at the following eight positions: Tyr32Glu, Leu65His, Ala67Gly, His70Gly, Phe108Tyr, Gln109His, Asp158Gly, and Leu162Gly, as described by Wang, 2006. We ordered the synthetase as gene synthesis and incorporated it via Gibson assembly in pSB1C3. For the experiments, the CDS for the synthetase needs to be on a low copy plasmid. Therefor we choose to insert it into pSB3C5 with a bio brick assembly.
To test whether the synthetase really incorporates CouAA, we transformed the synthetase in pSB3C5 and our composite part BBa_ K2201331 as test protein in E. coli BL21 DE3. The part K2201231 contains the CDS for the protein Sup35 with an amber stop codon at position 21 under control of a T7-promoter. If the synthetase works, it will incorporate CouAA in response to the amber codon at position 21. However, we expressed the test protein, which contains a histidine tag, and purified it through NiNTA chromatography. The purified proteins were analysed via UV/VIS-spectralphotometer, analysed on SDS-PAGE and digested with trypsin and analyzed through MALDI TOF/TOF.

Construction of RuBisCo mutants containing the amber stop codon

To incorporate the fluorescent amino acid at specific sites, we first had to choose the position and then use site directed mutagenesis to incorporate the amber stop codon at this position. For labeling issues, the fluorescent amino acid should not influence the protein folding or the activity. Furthermore, according to our expert Prof. Dr. Nedilijko Budisa, the yield of the protein could be influenced by the position the fluorescent amino acid is incorporated at. Therefore, we decided to try different positions at which the noncanonical amino acid is incorporated, all at permissive sites of the RuBisCo.
The permissive sites were detected by the alignment with Clustal Omega of different analogical RuBisCo from Thioalkalivibrio sulfidiphilus, Thioalkalivibrio denitrificans, Thiothrix nivea, Thermothiobacillus tepidarius, Acidobacillus caldus and Acidiferrobacter thiooxydans. If amino acids at the same position are heterologous, the amino acid seems to be unimportant for the functioning and folding of the protein. These permissive sites of the enzyme are suitable for the incorporation of the noncanonical fluorescent amino acid.

Figure 1: Multiple amino acid sequence alignment of analogical small subunit RuBisCo variants from Thioalkalivibrio sulfidiphilus(WP_012639733.1), Thioalkalivibrio denitrificans(WP_058575622.1), Thiothrix nivea(WP_012823800.1), Thermothiobacillus tepidarius(WP_066099403), Acidobacillus caldus(WP_002708379.1) and Acidiferrobacter thiooxydans(WP_045467882.1).

Due to the alignment, we decided to incorporate the fluorescent amino acid at position 2 and position 111 of the small subunit, and at position 474 of the large subunit. In addition, we decided to try different combinations of this positions to find out if this intensifies the fluorescence signal. Thus,we constructed the following seven RuBisCo mutants:

-BBa_K2201261 with a TAG at amino acid position 2 of the small subunit
-BBa_K2201262 with a TAG at amino acid position 111 of the small subunit
-BBa_K2201263 with a TAG at amino acid position 474 of the large subunit
-BBa_K2201264 with a TAG at amino acid position 2 and 111 of the small subunit
-BBa_K2201265 with a TAG at amino acid position 2 of the small subunit and amino acid position 474 of the large subunit
-BBa_K2201266 with a TAG at amino acid position 111 of the small subunit and amino acid position 474 of the large subunit
-BBa_K2201267 with a TAG at amino acid position 2 and 111 of the small subunit and amino acid position 474 of the large subunit

To compare the new tool using the noncanonical amino acid to the labeling with fluorescence proteins, we deigned the BBa_K2201620 containing a fusion of the CDS for the small subunit of the RuBisCo and the CDS for mRFP connected with a short linker.

Colocalization of the RuBisCO and the carboxysome

References

Wang, J., Xie, J., Schultz, P. G.(2006). A Genetically Encoded Fluorescent Amino Acid. American Chemical Society.128:8738-8739

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 <h2>Characterisation of the fluorescent amino acid <a href="https://2017.igem.org/wiki/index.php?title=Team:Bielefeld-CeBiTec/Project/toolbox/labeling#CouAA">L&#x2011;(7&#x2011;hydroxycoumarin&#x2011;4&#x2011;yl)&nbsp;ethylglycin (CouAA)</a></h2>
 <article>
-To find out if L&#x2011;(7&#x2011;hydroxycoumarin&#x2011;4&#x2011;yl)&nbsp;ethylglycin (CouAA) is suitable for cultivation and expression experiments we wanted to performe experiments on the stability and fluorescence ability. At first the absorbance of LB media containing 1 mM was measured at the beginning and after 24 hours incubation at 37 °C. It turns out that the absorbance maximum is at 328 nm and the loss of fluorescence is under 10 %, which can also be a result of the photobleaching of CouAA. After that we recorded a fluorescence spectra of the sample with constant irradiation at 328 nm. The fluorescence maximum was measured at 452 nm, both spectra are shown in figure 1. The absorbance and fluorescence spectra are very similar to the spectra published by Wang 2006. The small variances in the absorbance are probably caused by different absorptions of the used media. After incorporation in the protein we recorded the absorbance and fluorescence spectra of CouAA in the protein to, the spectra are shown in figure 3.
+To find out if L&#x2011;(7&#x2011;hydroxycoumarin&#x2011;4&#x2011;yl)&nbsp;ethylglycin (CouAA) is suitable for cultivation and expression experiments we performed experiments on the stability and fluorescence ability. At first the absorbance of LB media containing 1 mM was measured at the beginning and after 24 hours incubation at 37 °C. It turns out that the absorbance maximum is at 328 nm and the loss of fluorescence is under 10 %, which can also be a result of the photobleaching of CouAA. After that we recorded a fluorescence spectra of the sample with constant irradiation at 328 nm. The fluorescence maximum was measured at 452 nm, both spectra are shown in figure 1. The absorbance and fluorescence spectra are very similar to the spectra published by Wang 2006. The small variances in the absorbance are probably caused by different absorptions of the used media. After incorporation in the protein we recorded the absorbance and fluorescence spectra of CouAA in the protein to, the spectra are shown in figure 3.
 </article>