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		+	'''Cloning strategy'''
		+	Since our assembly construct with two insertion sites (LINK) was not ready at this point, we choose to follow a fast track cloning strategy to initiate trans- splicing of the split sfGFP. Therefore, we cloned the N-terminal protein part in front of the N- partial split intein NpuDnaE and the C-terminal GFP part (65-225) downstram of the C- portion of the intein using CPEC. The N- and C-terminal parts of GFP were cloned in a bicistronic expression backbone to allow expression via standard biobrick cloning. Also, non-splicing variants were generated by changing the essential cysteine at the N-terminal part of the N-intein to glycine (CG) and the serine and asparagine in the C-intein to alanine and glycine (SAAG). In order to splice, the inteins need a cysteine at the amino acid position where the splicing reaction will occur. Therefore the splicing product, in this case will harbour a cysteine at amino acid position 65. To ensure that the inserted cystein does not interfere with the fluorescence of the reconstituted sfGFP, we cloned a positive control of sfGFP by insertion of the cystein using mutagenesis PCR.
		+
		+	'''Fluorescence measurement'''
		+
		+	We expressed the split sfGFP construct with the various controls in BL21(DE3). 5ml cultures were inoculated with 500 µl of an overnight culture and induced with 1mM as soon as the cultures reached the OD600 of 0.8. The cultures incubated at 37 °C for further 4 hours. Subsequently the fluorescence was measured in a TECAN plate reader and the FACS. To ascertain the best excitation and emission wavelength in the plate reader we tested several excitation and emission wavelengths. The excitation at 475nm and emission at 512nm turned out to be most suitable for sfGFP. To validate our results we conducted several assays with a series of biological replicates following the same experimental layout. 300 µl of each sample were taken to measure the fluorescence in the plate reader on a black 96 well plate and a 1:1000 dilution of the original culture was prepared for the FACS measurements. The samples were loaded as described in the illustration below. Besides the in vivo expression and splicing reaction, also a mixture of N- and C-terminal split GFP-NpuDnaE in vitro was scheduled. In order to conduct a Western Blot the cells were harvested by centrifugation for 5 min at 3270 rcf at 4 °C. Subsequently they were re-suspended in 500 µl PBS on ice and sonicated for 2 min at 50% power on ice. The samples were centrifuged at 18000rcf for 10 min and the supernatant was re-suspended in 500 µl PBS on ice.  For the Western Blot and Coomassie Gels, the samples were mixed with 100 µl 5x Lämmli-Buffer and 10 µl were loaded on the SDS-Gel.
	[[{{PAGENAME}}/phototox|conditions testing]]		[[{{PAGENAME}}/phototox|conditions testing]]
	= Results =		= Results =
		+
		+	The CPEC cloning method seems to be a straightforward strategy to assemble several parts simultaneously. However CPEC with more than two inserts and one backbone hit on problems on the beginning of our project. Therefore our assembly construct with two insertion sides was not ready at the time point when we started with our fast track sfGFP assay to verify trans- splicing of NpuDnaE and the restoration of a protein’s functionality upon splicing. We decided to prove the fusion and activation of proteins with a fast track sfGFP assay, which we designed using CPEC cloning with only two inserts. The two inserts for the N-terminal part resulted in sfGFPN that includes amino acid 1 to 65 and the split intein NpuDnaEN, altogether reaching a size of 940 bp. Respectively, the C-terminal construct consists of NpuDnaEC-sfGFPc (65-225) with a size of 1074 bp. Subsequently the constructs were cloned on the same plasmid to allow bicistronic expression.
	= Discussion =		= Discussion =

---

Revision as of 08:34, 17 October 2014

Contents 1 Introduction 2 Methods 3 Results 4 Discussion 5 Outlook 5.1 References

Introduction

A common strategy for investigating molecular and cellular biological questions is the use of fusion proteins and to control the functions of these proteins in a spatial and temporal manner. Instead of manipulating proteins on the genomic level, we aimed at editing proteins post-translationally. By using our intein toolbox, one is able to fuse proteins and/or protein tags(Link), as well as reconstitute the function by complementing two formerly split halves of a single protein, and thereby recreate the function of the protein. Mechanistically, the reconstitution of split proteins is identical with the fusion of different proteins to their tags.

To demonstrate the restoration of function of a formerly split protein, we choose a set of fluorescent proteins, whose function can, when properly reassembled, easily be read out using their florescence. Split fluorescent proteins are rarely used in the context of intein splicing. However they are widely applied in bimolecular fluorescence complementation (BiFC) assays [1]. This approach is based on the complementation between fragments of fluorescent proteins that reconstitute its fluorescence when brought into proximity by associated interacting proteins.

Methods

Selection of Split Sites

For each of the fluorescent proteins, we selected split sites according to previous published research. Additionally to fluorescent proteins we choose to create two versions of split luciferases. A list of all designed constructs are depicted in the following table:

Protein	Split site	Comment
mRFP	154/155	Split between β - barrel 7 and 8 [2]
mCherry	168/169	Split between β - barrel 8 and 9 [3]
GFP	157/158	Split between barrel β - 7 and 8 [4]
sfGFP	64/65	In front of the chromphore region [5]
Firefly Luciferase	437/438	In flexible tether between the two subunits [6]
Renilla Luciferase	229/230	split between barrel β - 7 and 8 [7]

Cloning strategy

Since our assembly construct with two insertion sites (LINK) was not ready at this point, we choose to follow a fast track cloning strategy to initiate trans- splicing of the split sfGFP. Therefore, we cloned the N-terminal protein part in front of the N- partial split intein NpuDnaE and the C-terminal GFP part (65-225) downstram of the C- portion of the intein using CPEC. The N- and C-terminal parts of GFP were cloned in a bicistronic expression backbone to allow expression via standard biobrick cloning. Also, non-splicing variants were generated by changing the essential cysteine at the N-terminal part of the N-intein to glycine (CG) and the serine and asparagine in the C-intein to alanine and glycine (SAAG). In order to splice, the inteins need a cysteine at the amino acid position where the splicing reaction will occur. Therefore the splicing product, in this case will harbour a cysteine at amino acid position 65. To ensure that the inserted cystein does not interfere with the fluorescence of the reconstituted sfGFP, we cloned a positive control of sfGFP by insertion of the cystein using mutagenesis PCR.

Fluorescence measurement

We expressed the split sfGFP construct with the various controls in BL21(DE3). 5ml cultures were inoculated with 500 µl of an overnight culture and induced with 1mM as soon as the cultures reached the OD600 of 0.8. The cultures incubated at 37 °C for further 4 hours. Subsequently the fluorescence was measured in a TECAN plate reader and the FACS. To ascertain the best excitation and emission wavelength in the plate reader we tested several excitation and emission wavelengths. The excitation at 475nm and emission at 512nm turned out to be most suitable for sfGFP. To validate our results we conducted several assays with a series of biological replicates following the same experimental layout. 300 µl of each sample were taken to measure the fluorescence in the plate reader on a black 96 well plate and a 1:1000 dilution of the original culture was prepared for the FACS measurements. The samples were loaded as described in the illustration below. Besides the in vivo expression and splicing reaction, also a mixture of N- and C-terminal split GFP-NpuDnaE in vitro was scheduled. In order to conduct a Western Blot the cells were harvested by centrifugation for 5 min at 3270 rcf at 4 °C. Subsequently they were re-suspended in 500 µl PBS on ice and sonicated for 2 min at 50% power on ice. The samples were centrifuged at 18000rcf for 10 min and the supernatant was re-suspended in 500 µl PBS on ice. For the Western Blot and Coomassie Gels, the samples were mixed with 100 µl 5x Lämmli-Buffer and 10 µl were loaded on the SDS-Gel.

conditions testing

Results

The CPEC cloning method seems to be a straightforward strategy to assemble several parts simultaneously. However CPEC with more than two inserts and one backbone hit on problems on the beginning of our project. Therefore our assembly construct with two insertion sides was not ready at the time point when we started with our fast track sfGFP assay to verify trans- splicing of NpuDnaE and the restoration of a protein’s functionality upon splicing. We decided to prove the fusion and activation of proteins with a fast track sfGFP assay, which we designed using CPEC cloning with only two inserts. The two inserts for the N-terminal part resulted in sfGFPN that includes amino acid 1 to 65 and the split intein NpuDnaEN, altogether reaching a size of 940 bp. Respectively, the C-terminal construct consists of NpuDnaEC-sfGFPc (65-225) with a size of 1074 bp. Subsequently the constructs were cloned on the same plasmid to allow bicistronic expression.

Discussion

Outlook

References

[1] Hu, C-D, & T.,K., Kerppola. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol. 21(5), 539-545 (2003).

[2] Jach, G., Pesch, M., Richter, K., Frings, S., & Uhrig, J., F. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nature Methods, 3, 597-600 (2006).

[3] Furman, J., L., Badran, A., H., Shen, S., Stains, C., I., Hannallah, J., Segal, D., J., Ghosh, I. Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA. Bioorg Med Chem Lett, 19(14), 3748-3751 (2009).

[4] Oyawa, T., Takeuchi, M., Kaihara, A., Sato, M., Umezawa, Y. Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem, 73, 5866-5874 (2001).

[5] Aranko, A., S., Oeemig, J., S., Kajander, T., & Iwai, H. Intermolecular domain swapping induces intein-mediated protein alternative splicing. Nat. Chem. Bio., 9, 616-622 (2013).

[6] Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y. Split luciferase as an optical probe for detecting protein- protein interactions in mammalian cells based on protein splicing. Anal. Chem, 73, 2516-2521 (2001).

[7] Kim, S. B., Ozawa, T., Watanabe, S., Umezawa, Y. High- throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split renilla luciferase. PNAS, 101, 11542–11547 (2004).