Parts

On this page, you can find the parts we added to the registry for further usage, by future iGEM teams and synthetic biologists all over the world. We present the parts required for the expression of ferritin, for including the cell-penetrating peptides, for testing their penetration efficiency, and the parts needed for introducing the non-canonical amino acid to ferritin. Furthermore, we have listed all composite parts that we have constructed and briefly explained their function.

If you need more details or if you have questions regarding any of our parts, feel free to contact us, referring to the team of 2023. We are more than happy to help!

Basic Parts

Name	Type	Short Description	Description	Designers	Length
BBa_K4669000	Coding	WT-Ferritin	Coding for one subunit of the wildtype human heavy chain ferritin protein. 24 subunits assemble to one container.	Prof. Dr. Tobias Beck	552 bp
BBa_K4669001	Coding	R9	Coding for the R9 cell-penetrating peptide, consisting of nine arginines	Dr. Dirk Becker	27 bp
BBa_K4669002	Coding	R12	Coding for the R12 cell-penetrating peptide, consisting of twelve arginines	Dr. Dirk Becker	42 bp
BBa_K4669003	Coding	GS-Linker	Coding for a glycine-serine-linker, used for enabling correct folding of two fused peptide chains.	Dr. Dirk Becker	15 bp
BBa_K4669007	Coding	Ferritin K88TAG	Coding for the wildtype human heavy chain ferritin. Mutation: K88TAG (Amber codon)	Lisa Siemers	552 bp
BBa_K4669011	Coding	Mm_NESPylRS	Coding for a aminoacyl-tRNA synthetase derived from Methanosarcina mazei. Used for loading the Pyl-tRNA	Das, Dibyendu Kumar et al. ^[1]	1392 bp
BBa_K4669012	Terminator	rrnB Terminator	rrnB Terminator to terminate expression	Chin et al. ^[2]	158 bp
BBa_K4669013	Regulatory	araBAD Promoter	promoter inducible by arabinose	Chin et al. ^[2]	285 bp
BBa_K4669015	Coding	eGFP	Coding for the enhanced green fluorescent protein	Dr. Dirk Becker	708 bp
BBa_K4669016	Coding	TAT2	Coding for the TAT2 cell-penetrating peptide, consisting of two TAT peptides.	Dr. Dirk Becker	54 bp
BBa_K4669017	Coding	TAT3	Coding for the TAT3 cell-penetrating peptide, consisting of three TAT peptides.	Dr. Dirk Becker	99 bp
BBa_K4669018	Coding	TAT-R9	Coding for the TAT-R9 cell-penetrating peptide, consisting of one TAT and one R9 peptide.	Dr. Dirk Becker	60 bp
BBa_K4669019	Coding	HA-Tag	HA-Tag, used for enabling correct folding of two fused peptide chains	Dr. Dirk Becker	27 bp
BBa_K4669020	Plasmid backbone	pET22b(+)	pET22b(+) plasmid backbone for IPTG induced expression in E. coli	Prof. Dr. Tobias Beck	5379 bp

Composite Parts

Name	Type	Short Description	Description	Designers	Length
BBa_K4669004	Coding	R9-Ferritin	The cell-penetrating peptide R9 fused to WT-ferritin	Lisa Siemers	597 bp
BBa_K4669005	Coding	TAT-Ferritin	The cell-penetrating peptide TAT fused to WT-ferritin	Lisa Siemers	603 bp
BBa_K4669006	Coding	R12-Ferritin	The cell-penetrating peptide R12 fused to WT-ferritin	Lisa Siemers	612 bp
BBa_K4669008	Coding	TAT-Ferritin (Amber)	The cell-penetrating peptide TAT fused to ferritin with the mutation K88TAG	Lisa Siemers	603 bp
BBa_K4669009	Coding	R9-Ferritin (Amber)	The cell-penetrating peptide R9 fused to ferritin with the mutation K88TAG	Lisa Siemers	597 bp
BBa_K4669010	Coding	R12-Ferritin (Amber)	The cell-penetrating peptide R12 fused to ferritin with the mutation K88TAG	Lisa Siemers	612 bp
BBa_K4669014	Coding	aaRS/tRNA expression system	The expression for the PylRS and two copies of the Pyl tRNA. Inducible by arabinose.	Jonas Westphal, Lisa Siemers	2791 bp
BBa_K4669021	Coding	R9-eGFP	The cell-penetrating peptide R9 fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	810 bp
BBa_K4669022	Coding	R12-eGFP	The cell-penetrating peptide R12 fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	822 bp
BBa_K4669023	Coding	TAT-eGFP	The cell-penetrating peptide TAT fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	813 bp
BBa_K4669024	Coding	TAT2-eGFP	The cell-penetrating peptide TAT2 fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	834 bp
BBa_K4669025	Coding	TAT3-eGFP	The cell-penetrating peptide TAT3 fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	879 bp
BBa_K4669026	Coding	TAT-R9-eGFP	The cell-penetrating peptide TAT-R9 fused to enhanced green fluorescent protein (eGFP)	Dr. Dirk Becker	840 bp
BBa_K4669027	Plasmid	pET22b(+)_WT-Ferritin	The complete plasmid we used for the expression of WT-ferritin and all further experiments with ferritin. Restriction recognition sites for SapI and BsaI were deleted.	Prof. Dr. Tobias Beck, Lisa Siemers, Maren Hinz	5931 bp
BBa_K4669028	Plasmid	pET22b(+)_TAT-Ferritin	The complete plasmid we used for the expression of TAT-ferritin and all further experiments with ferritin. Restriction recognition sites for SapI and BsaI were deleted.	Lisa Siemers	5979 bp
BBa_K4669029	Plasmid	pET22b(+)_R9-Ferritin	The complete plasmid we used for the expression of R9-ferritin and all further experiments with ferritin. Restriction recognition sites for SapI and BsaI were deleted.	Lisa Siemers	5995 bp
BBa_K4669030	Plasmid	pET22b(+)_R12-Ferritin	The complete plasmid we used for the expression of R12-ferritin and all further experiments with ferritin. Restriction recognition sites for SapI and BsaI were deleted.	Lisa Siemers	6010 bp

Nomination Best Composite Part

Introducing it to iGEM, we would like to nominate our plasmid pET22b(+)_WT-Ferritin (BBa_K4669027) (fig. 1). This plasmid, which integrates a ready-to-use construct for expressing the wildtype humane heavy chain ferritin (BBa_K4669000) in Escherichia coli, was derived from a plasmid of Prof. Dr. Tobias Beck.

Failed to load image — Fig. 1: Plasmid map for our composite part BBa_K4669027. WT-Ferritin, introduced in a pET22b(+) backbone, which we mutated to delete SapI and BsaI resitriction recognition sites. The backbone carries a ampicillin resistance and T7 promoters, inducible by adding IPTG.

For contributing the full plasmid to iGEM, we have performed mutagenesis to delete restriction recognition sites for the enzymes BsaI and SapI. Thus, we established a plasmid, compatible with the iGEM regulations and are able to provide the plasmid to future iGEM teams for directly diving into cloning and expression experiments!

The wildtype ferritin displays the heart of our project and of our modularity: New cell-penetrating peptides (CPPs) can be fused to the N-terminus by using a simple PCR for mutagenesis. An Amber codon can be introduced to the plasmid at a wished site by simple QuikChange mutagenesis and, as an outlook, amino acids can be mutated to alter the interactions of the ferritin with its environment or its cargo.

Furthermore, we have successfully conducted experiments on amplifying the plasmid and transforming it into the E. coli strain BL21 (DE3) star for expression. We were able to show that our expression was fruitful. Afterwards we were capable to completely purify the ferritin in an ion exchange chromatography (IEC), followed by two rounds of size exclusion chromatography (SEC). With our purified ferritin container we performed negative staining using Transmission Electron Microscopy (TEM). Fore more information about all of these accomplishments, we invite you to have a look at our results page!