Parts

This page outlines the standard biological parts used by the iGEM team Tuebingen 2021, thereby focusing on their function and interplay.

We used a great number of parts in our iGEM project: Eight new basic parts and four new composite parts were added to the registry. Furthermore, we used seven already registered parts .

On this page, we listed our parts, elaborated their biology and their usage in our iGEM project. For full documentation and information on the results we obtained for the parts, please visit the Registry of standard biological parts , or our results page .

Table 1 summarizes and categorizes all the biological parts created by our team. Table 2 lists the biological parts from the Registry used by our team.

Table 1: New parts created by our team.

Name	Type	Description	Designer	Length (bp)
BBa_K3757000	Coding	CtAEP1	Catia Harnack	1450
BBa_K3757001	Coding	Oak1 precursor	Erik Zimmer	999
BBa_K3757002	Coding	c_blank	Catia Harnack	142
BBa_K3757003	Coding	c_CHEN_1	Catia Harnack	175
BBa_K3757004	Coding	c_CHEN_6	Erik Zimmer	161
BBa_K3757005	Coding	c_KR-12_1	Erik Zimmer	164
BBa_K3757006	Coding	c_KR-12_6	Erik Zimmer	164
BBa_K3757007	Composite	35S Oak1	Erik Zimmer	2117
BBa_K3757008	Tag	c-myc tag, C-terminal	Erik Zimmer	30
BBa_K3757009	Composite	35S sGFP (S65T)	Erik Zimmer	1805
BBa_K3757010	Composite	35S CtAEP1	Erik Zimmer	2565
BBa_K3757011	Composite	3in1 Oak1 CtAEP1 sGFP (S65T)	Erik Zimmer	6805

Table 1: Parts from the Registry used by our team.

Name	Type	Description	Length (bp)
BBa_K1159307	Terminator	35S Terminator of Cauliflower Mosaic Virus (CaMV)	217
BBa_K3183024	Tag	HA Tag for L.reuteri	27
BBa_K3669012	Reporter	sGFP (S65T)	720
BBa_K233327	DNA	Agrobacterium tumefaciens Right Border Consensus Sequence	25
BBa_K233326	DNA	Agrobacterium tumefaciens Left Border Consensus Sequence	25
BBa_K788000	Regulatory	CamV 35s promoter for plant expression	835
BBa_M50428	Tag	6x His tag	18

Go To

(BBa_K3757000) CtAEP1 (BBa_K3757001) Oak1 precursor (BBa_K3757002) c_blank (BBa_K3757003) c_CHEN_1 (BBa_K3757004) c_CHEN_6 (BBa_K3757005) c_KR-12_1 (BBa_K3757006) c_KR-12_6 (BBa_K3757008) c-myc tag (BBa_K3183024) HA-tag (BBa_M50428) His6-tag (BBa_K233326) <i>A. tumefaciens</i> LB (BBa_K233327) <i>A. tumefaciens</i> RB (BBa_K788000) CaMV 35S promoter (BBa_K1159307) 35S terminator (BBa_K3669012) sGFP S65T

New Parts

We created eight new basic parts in our iGEM project. They are mostly involved in the biosynthesis of cyclic, stabilized antimicrobial peptides (AMPs) in Nicotiana benthamiana ( N. benthamiana ) when co-expressed from our 3in1 composite part ( BBa_K3757011 ). Therefore, the codon-usage of these parts is optimized for expression in plants. The interplay between these parts is illustrated in the figure below.

The cyclization process of cyclotides in plants. — Figure: Schematic illustration of the steps and parts involved in the biosynthesis of a cyclic cyclotide construct.

A grafted cyclotide construct is expressed in the Oak1 precursor peptide. This precursor is post-translationally processed in the ER and Golgi leading to the formation of the characteristic disulfide bridges in the cyclotide. Finally, the precusor translocates into the vacuole, where it is cyclized by the asparaginyl endopeptidase CtAEP1 .

In our iGEM project, we expressed AMPs in a cyclotide scaffold attempting to stabilize the AMPs. The part numbers, abbreviations and names of all the AMP-cyclotide constructs we worked with are shown in the table below.

Table: Overview of the grafted cyclotide constructs used by iGEM team Tuebingen 2021.

registry no.	short name	long name	3in1	2in1
BBa_K3757002	c_blank	MCoTI-II loop 5 His6	X
BBa_K3757003	c_CHEN_1	MCoTI-II loop 1 CHEN, loop 5 His6	X	X
BBa_K3757004	c_CHEN_6	MCoTI-II loop 6 CHEN, loop 5 His6	X	X
BBa_K3757005	c_KR-12_1	MCoTI-II loop 1 KR-12, loop 5 His6	X
BBa_K3757006	c_KR-12_6	MCoTI-II loop 6 KR-12, loop 5 His6	X

( BBa_K3757000 ) CtAEP1

Asparaginyl endopeptidase 1 with ligase activity from Clitoria ternatea . Can be used to cyclize peptides embedded in the Oak1 precursor.

CtAEP1, also called butelase 1, is an Asx-specific ligase from the family of asparaginyl endopeptidases (AEP) (EC 3.4.22.34). It originates from the herb Clitoria ternatea . Compared to other endopeptidases, it does not only cleave peptide bonds, but it ligates two cleaved termini. It therefore specifically recognizes substrates with the sequence “NHV” or “DHV” as a C-terminal propeptide. The sequence requirements for the N-terminal cleavage recognition site “CXG” are lower, requiring X not to be a proline residue. The peptide bond between N/D and HV is cleaved, linking the C-terminal N/D to the N-terminal G. With a catalytic efficiency of 542,000 M ^-1 s ^-1 , CtAEP1 is the fastest protein ligase known. Its natural function is the cyclization of cyclotides , which are peptides involved in plants’ defense mechanism. ¹ CtAEP1 is expressed as an inactive precursor protein with a C-terminal cap-domain blocking its active site. After translation, it is localized to the vacuole by its N-terminal signal sequence. The low pH environment in the vacuole leads to autocatalytic cleavage of the cap-domain exposing the catalytic triad and thereby activating it. Cyclization and therefore activation of its substrate cyclotides takes place in the vacuole. ²

CtAEP1 has been successfully used for the cyclization of proteins in vitro ³ . It was also demonstrated that CtAEP1 could lead to cyclization of cyclotides in Nicotiana benthamiana ( N. benthamiana ), when both the cyclotide precursor and the CtAEP1 were recombinantly coexpressed ⁴ .

iGEM team Tuebingen 2021 used CtAEP1 to express stabilized antimicrobial peptides (AMPs) in N. benthamiana . These peptides are grafted into a cyclotide scaffold, which can be backbone cyclized by the co-expressed CtAEP1. For further information, visit the project description wiki page of iGEM Tuebingen 2021 .

CtAEP1 was expressed in N. benthamiana , regulated by a CaMV 35S promoter ( BBa_K788000 ) and a 35S terminator ( BBa_K1159307 ), C-terminally tagged with a HA-tag ( BBa_K3183024 ). This is summarized as the composite part BBa_K3757010 . In 3in1 vectors ( BBa_K3757011 ), the AEP was co-expressed with cyclotide constructs embedded in the Oak1 precursor ( BBa_K3757001 ), and a GFP reporter gene ( BBa_K3669012 ). Furthermore, 2in1 vectors were cloned, which contain only the genes for the Oak1 precursor and GFP, but not for CtAEP1. For further information, visit the experiments wiki page of iGEM Tuebingen 2021 .

References

Nguyen, G. K. T [Giang K. T.], Wang, S [Shujing], Qiu, Y., Hemu, X., Lian, Y., & Tam, J. P [James P.] (2014). Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nature Chemical Biology, 10(9), 732–738. https://doi.org/10.1038/nchembio.1586
James, A. M., Haywood, J., Leroux, J., Ignasiak, K., Elliott, A. G., Schmidberger, J. W., Fisher, M. F., Nonis, S. G., Fenske, R., Bond, C. S., & Mylne, J. S. (2019). The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity. The Plant Journal : For Cell and Molecular Biology, 98(6), 988–999. https://doi.org/10.1111/tpj.14293
Nguyen, G. K. T [Giang K. T.], Kam, A., Loo, S., Jansson, A. E., Pan, L. X., & Tam, J. P [James P.] (2015). Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization. Journal of the American Chemical Society, 137(49), 15398–15401. https://doi.org/10.1021/jacs.5b11014
Poon, S., Harris, K. S., Jackson, M. A., McCorkelle, O. C., Gilding, E. K., Durek, T., van der Weerden, N. L., Craik, D. J [David J.], & Anderson, M. A. (2018). Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. Journal of Experimental Botany, 69(3), 633–641. https://doi.org/10.1093/jxb/erx422

( BBa_K3757001 ) Oak1 precursor

Cyclotide kalata B1 precursor peptide from Oldenlandia affinis can be used to express cyclic peptides embedded within the precursor in Nicotiana benthamiana, when co-expressed with CtAEP1. Sequences of these peptides can be introduced into the precursor in a single cloning step, using the Type IIS restriction enzyme Esp3I and blue-white selection.

Oak1 is the precursor protein of the cyclotide kalata B1 from the herb Oldenlandia affinis . Cyclotides are cyclic peptides involved in plant host defense. The native Oak1 precursor harbors the sequence encoding the actual cyclotide, flanked by a N-terminal and a C-terminal propeptide . ¹ It also contains an '''ER-signal sequence''' at its N-terminus, which is cleaved upon localization of the precursor to the ER. In the ER, three disulfide bonds are formed, building the cyclic cystine knot motif, which is characteristic for cyclotides. Afterward, the Oak1 precursor translocates to the vacuole where it is cyclized by the cyclizing asparaginyl endopeptidase (AEP) OaAEP1 and thereby activated. The AEP enzyme recognizes a specific amino acid sequences in the N- and C-terminal propeptides, leading to subsequent cleavage of the propeptides and cyclization of kalata B1. ²

iGEM team Tuebingen 2021 used the Oak1 precursor to express stabilized antimicrobial peptides (AMPs) in a cyclotide scaffold.

Several adjustments were made to the native Oak1 sequence:

Team Tuebingen used the AEP CtAEP1 ( BBa_K3757000 ) for cyclization of the cyclotides, which has a very high catalytic efficiency. Compared to OaAEP1, CtAEP1 has different recognition sequence requirements for the cyclotide precursor ³ . The C-terminal propeptide was therefore removed and replaced by a sequence encoding the dipeptide “HV”.
The sequence encoding kalata B1 cyclotide was replaced by a ''' lacZ operon'''. This is composed of lac promoter, lac operator and the lacZ gene encoding β-galactosidase. The operon is flanked by Esp 3I type IIS restriction sites allowing scarless replacement of the lacZ operon with an arbitrary DNA sequence in a single Golden Gate cloning step. Introduction of new AMP-encoding sequences into the Oak1 precursor should therefore be easy and efficiently facilitated. Success of this cloning step can be monitored by blue-white selection.

The modified Oak1 precursor was expressed in N. benthamiana , regulated by a CaMV 35S promoter ( BBa_K788000 ) and a 35S terminator ( BBa_K1159307 ), C-terminally tagged with a c-myc-tag ( BBa_K3757008 ). This is summarized as the composite part BBa_K3757007 . This composite part was cloned into a vector with the genes encoding CtAEP1 ( BBa_K3757001 ) and GFP ( BBa_K3669012 ) as a reporter gene, forming a 3in1 vector ( BBa_K3757011 ). Also, as a control, a 2in1 vector was constructed, which misses the CtAEP1 encoding gene. In these 3in1 and 2in1 vectors, an arbitrary AMP construct may be inserted in a single cloning step. The resulting vector can then be used for transient transfection of N. benthamiana leaves to express the stabilized AMPs.

References

Poon, S., Harris, K. S., Jackson, M. A., McCorkelle, O. C., Gilding, E. K., Durek, T., van der Weerden, N. L., Craik, D. J., & Anderson, M. A. (2018). Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. Journal of Experimental Botany, 69(3), 633–641. https://doi.org/10.1093/jxb/erx422
Narayani, M., Babu, R., Chadha, A., & Srivastava, S. (2020). Production of bioactive cyclotides: a comprehensive overview. Phytochemistry Reviews, 19(4), 787–825. https://doi.org/10.1007/s11101-020-09682-9
Nguyen, G. K. T [Giang K. T.], Wang, S [Shujing], Qiu, Y., Hemu, X., Lian, Y., & Tam, J. P [James P.] (2014). Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nature Chemical Biology, 10(9), 732–738. https://doi.org/10.1038/nchembio.1586

The Cyclotide Scaffold

MCoTI-II is a squash trypsin inhibitor cyclotide, also known as cyclic knottins, and originates from the plant species Momordica cochinchinensis ¹ . Cyclotides are a class of plant cyclic peptides with a length of around 30 amino acids (aa). They comprise three disulfide bridges, which together with their cyclic backbone form the characteristic cyclic cystine knot motif making the cyclotide structure very rigid. This motif is responsible for cyclotides’ exceptional stability towards proteases and heat. The regions between their six cysteine residues are called loops 1 to 6. Cyclotides are classified into three categories by their sequence: Möbius, bracelet and trypsin inhibitor cyclotides with the latter having a low sequence conservation compared to the other two classes. ² In plants, these peptides are expressed as precursors, which are finally localized to the vacuole. There, the actual cyclotide sequences are recognized by specialized cyclizing asparaginyl endopeptidases (AEPs), which are catalyzing the backbone cyclization. ³ MCoTI-II itself is 34 aa in length, with the sequence GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD , and is folded into a cystine stabilized triple-stranded beta-sheet. Its natural function as a trypsin inhibitor is based on its inhibitory loop 1. Thereby, it acts as a defensive agent against the plant’s predators. ¹ MCoTI-II does not disrupt cell membranes and does not display antibacterial, nor hemolytic activity. ⁴

Cyclotides like MCoTI-II have been used for grafting approaches in the past. Thereby, grafting means the insertion into or the replacement of one of the cyclotide’s loops with another peptide sequence. This enables combining the cyclotide’s stability with the inserted peptide’s biological activity ⁴ . Regarding MCoTI-II, successful grafting into loops 1, 3, 5 and 6 has been demonstrated ⁵ .

This biological part is a grafted construct of MCoTI-II designed and tested by iGEM team Tuebingen 2021. Two different antimicrobial peptides (AMPs) were used and either grafted into loop 1 or loop 6 of the cyclotide. In addition, a His6-tag ( BBa_M50428 ) was grafted into loop 5 for affinity purification and immunodetection. Furthermore, flexible GS-linkers were included at the interface of the cyclotide’s native sequence and the grafted peptide.

The Grafted AMPs

CHEN is a modified peptide fragment of the AMP chensinin-1b, which itself is a strongly modified analogue of the naturally occurring 18 aa long AMP chensinin-1. Chensinin-1 occurs on the skin of the frog species Rana chensinensis and is active against gram-positive bacteria. The 18 aa long analogue chensinin-1b is active against both gram-positive and gram-negative bacteria. Its NMR structure displays a central alpha-helical region and an N-terminal random-coil region. To optimize the antimicrobial properties of chensinin-1b, the truncated and modified peptide [R ⁴ ,R ¹⁰ ]C1b(3–13) – hereafter called CHEN – was created. CHEN is 11 aa long and has the sequence VWRRWRRFWRR . The alternating hydrophobic (V, W) and cationic (R) residues lead to an amphipathic character in its alpha-helical fold, with a hydrophobic and a cationic face of the helix. This fold was shown to be essential for the antibacterial activity of CHEN, which involves the fast disruption of cell membranes. CHEN shows minimal inhibitory concentrations (MIC) of 1.56 μM to 3.13 μM against different clinically relevant gram-positive and gram-negative bacterial species, making it a potent antibacterial agent. Furthermore, it does not exhibit hemolytic activity up to a concentration of 500 μM. ⁶

KR-12 is the smallest antibacterial fragment of the human cathelicidin LL-37 ( BBa_K245114 ), which has been used by many iGEM teams in the past. LL-37 is an AMP with 37 aa and an alpha-helical secondary structure. It has potent immunomodulatory and antimicrobial properties and can promote wound healing. Furthermore, LL-37 displays cytotoxicity in the micromolar range. The 12 aa long fragment KR-12 is located nearby LL-37’s C-terminus and has the sequence KRIVQRIKDFLR . It also folds into an amphipathic alpha-helix with a cationic and a hydrophobic face. This structural feature is necessary for the peptide’s mode of action of cell membrane disruption by the carpet-model. KR-12 was demonstrated to be a potent antibacterial agent with minimal inhibitory concentrations (MIC) of 2.5 μM to 10 μM against different clinically relevant gram-positive and gram-negative bacterial species. In addition, it does not exhibit hemolytic activity up to a concentration of 80 μM. ⁷

The design

The grafted construct was cloned into an Oak1 precursor peptide ( BBa_K3757007 ) and expressed in N. benthamiana , regulated by a CaMV 35S promoter ( BBa_K788000 ) and a 35S terminator ( BBa_K1159307 ), and C-terminally tagged with a c-myc-tag ( BBa_K3757008 ). This is summarized as the composite part BBa_K3757007 . This composite part was cloned into a vector with the genes encoding CtAEP1 ( BBa_K3757001 ) and GFP ( BBa_K3669012 ) as a reporter gene, forming a 3in1 vector ( BBa_K3757011 ). Also, as a control, a 2in1 vector was constructed, which misses the CtAEP1 encoding gene. The resulting vector can then be used for transient transfection of N. benthamiana leaves to express the stabilized AMPs.

( BBa_K3757002 ) c_blank

This construct c_blank is MCoTI-II with a His6-tag grafted into loop 5.

Sequence and features of the construct c_blank. — Peptide sequence and features of the cyclotide grafting construct c_blank.

( BBa_K3757003 ) c_CHEN_1

This construct c_CHEN_1 is MCoTI-II with the AMP CHEN grafted into loop 1, as well as a His6-tag grafted into loop 5.

Sequence and features of the construct c_CHEN_1. — Peptide sequence and features of the cyclotide grafting construct c_CHEN_1.

( BBa_K3757004 ) c_CHEN_6

This construct c_CHEN_6 is MCoTI-II with the AMP CHEN grafted into loop 6, as well as a His6-tag grafted into loop 5.

Sequence and features of the construct c_CHEN_6. — Peptide sequence and features of the cyclotide grafting construct c_CHEN_6.

( BBa_K3757005 ) c_KR-12_1

This construct c_KR-12_1 is MCoTI-II with the AMP KR-12 grafted into loop 1, as well as a His6-tag grafted into loop 5.

Sequence and features of the construct c_KR-12_1. — Peptide sequence and features of the cyclotide grafting construct c_KR-12_1.

( BBa_K3757006 ) c_KR-12_6

This construct c_KR-12_1 is MCoTI-II with the AMP KR-12 grafted into loop 1, as well as a His6-tag grafted into loop 5.

Sequence and features of the construct c_KR-12_6. — Peptide sequence and features of the cyclotide grafting construct c_KR-12_6.

References

Heitz, A., Hernandez, J. F., Gagnon, J., Hong, T. T., Pham, T. T., Nguyen, T. M., Le-Nguyen, D., & Chiche, L. (2001). Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins. Biochemistry, 40(27), 7973–7983. https://doi.org/10.1021/bi0106639
Craik, D. J., & Conibear, A. C. (2011). The chemistry of cyclotides. The Journal of Organic Chemistry, 76(12), 4805–4817. https://doi.org/10.1021/jo200520v
Conlan, B. F., Gillon, A. D., Barbeta, B. L., & Anderson, M. A. (2011). Subcellular targeting and biosynthesis of cyclotides in plant cells. American Journal of Botany, 98(12), 2018–2026. https://doi.org/10.3732/ajb.1100382
Koehbach, J., Gani, J., Hilpert, K., & Craik, D. J. (2021). Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens. Microorganisms, 9(6), 1249. https://doi.org/10.3390/microorganisms9061249
Craik, D. J., & Du, J. (2017). Cyclotides as drug design scaffolds. Current Opinion in Chemical Biology, 38, 8–16. https://doi.org/10.1016/j.cbpa.2017.01.018
Dong, W., Dong, Z., Mao, X., Sun, Y., Li, F., & Shang, D. (2016). Structure-activity analysis and biological studies of chensinin-1b analogues. Acta Biomaterialia, 37, 59–68. https://doi.org/10.1016/j.actbio.2016.04.003
Gunasekera, S., Muhammad, T., Strömstedt, A. A., Rosengren, K. J., & Göransson, U. (2020). Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability. Frontiers in Microbiology, 11, 168. https://doi.org/10.3389/fmicb.2020.00168

( BBa_K3757008 ) c-myc-tag

The c-myc-tag 10 aa long affinity tag, which is widely used on recombinant proteins ¹ . We tagged our Oak1 precursor ( BBa_K3757001 ) C-terminally with the c-myc-tag in the composite part 35S Oak1 ( BBa_K3757007 ). Even though we didn't use the tag for affinity purification or immunodetection, it has great potential to get characterised by future iGEM teams as a potent affinity tag.

References

Dennler, P., Bailey, L. K., Spycher, P. R., Schibli, R., & Fischer, E. (2015). Microbial transglutaminase and c-myc-tag: A strong couple for the functionalization of antibody-like protein scaffolds from discovery platforms. Chembiochem : A European Journal of Chemical Biology, 16(5), 861–867. https://doi.org/10.1002/cbic.201500009

Parts from the Registry

We used seven basic parts from the Registry in our composite parts.

( BBa_K3183024 ) HA-tag

The HA-tag is a 9 aa affinity tag derived from influenza viral glycoprotein hemagglutinin ¹ . We used this part in our composite part 35S CtAEP1 ( BBa_K3757010 ) to C-terminally tag the enzyme CtAEP1 ( BBa_K3757000 ). Thereby, we tried to detect CtAEP1 in plant crude extracts using an anti-HA-tag antibody.

References

Liu, D., Liu, C., Hu, J., Hang, L., Li, X., Wei, Y., Zhu, H., Zhang, Q., & Wang, X. (2018). Construction and evaluation of HA-epitope-tag introduction onto the VP1 structural protein of a novel HY12 enterovirus. Virology, 525, 106–116. https://doi.org/10.1016/j.virol.2018.09.010

( BBa_M50428 ) His6-tag

The His6-tag is widely used to tag proteins on either their N- or C-terminus. However, since we expressed cyclic peptides in our project, we tried to used the His6-tag internally in our peptide backbone, for affinity purification and for detection of the peptides with an anti-His-tag antibody. The His6-tag was implemented in all our grafted cyclotide constructs ( BBa_K3757002 , BBa_K3757003 , BBa_K3757004 , BBa_K3757005 , BBa_K3757006 ).

( BBa_K233326 ) A. tumefaciens LB

( BBa_K233327 ) A. tumefaciens RB

The left and the right border are sequences on the Ti-plasmid of Agrobacteria , which allow the transfer of the DNA between them, the T-DNA, into the plant cell, where it gets integrated into the plant's genome ¹ . We successfully used these two parts in our 3in1 expression vectors to transfer our composite part 3in1 Oak1 CtAEP1 sGFP (S65T) ( BBa_K3757011 ) into the genome of infiltrated N. benthamiana leaf cells.

References

Culianez-Macia, F. A., & Hepburn, A. G. (1988). Right-border sequences enable the left border of an Agrobacterium tumefaciens nopaline Ti-plasmid to produce single-stranded DNA. Plant Molecular Biology, 11(4), 389–399. https://doi.org/10.1007/BF00039019

( BBa_K788000 ) CaMV 35S promoter

The 35S promoter originating from cauliflower mosaic virus (CaMV) is a strong, constitutive promoter for gene expression in eukaryotes ¹ . We used this part for the expression of CtAEP1 ( BBa_K3757000 ), Oak1 precursor ( BBa_K3757001 ) and sGFP (S65T) ( BBa_K3757009 ) in the composite parts BBa_K3757010 , BBa_K3757007 and BBa_K3757009 , respectively.

References

Binder, A., Lambert, J., Morbitzer, R., Popp, C., Ott, T., Lahaye, T., & Parniske, M. (2014). A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PloS One, 9(2), e88218. https://doi.org/10.1371/journal.pone.0088218

( BBa_K1159307 ) 35S terminator

The 35S terminator is a polyadenylation signal for gene expression in eukaryotes ¹ . We used this part for the expression of CtAEP1 ( BBa_K3757000 ), Oak1 precursor ( BBa_K3757001 ) and sGFP (S65T) ( BBa_K3757009 ) in the composite parts BBa_K3757010 , BBa_K3757007 and BBa_K3757009 , respectively.

References

Binder, A., Lambert, J., Morbitzer, R., Popp, C., Ott, T., Lahaye, T., & Parniske, M. (2014). A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PloS One, 9(2), e88218. https://doi.org/10.1371/journal.pone.0088218

( BBa_K3669012 ) sGFP S65T

sGFP (S65T) is one of many mutants of the green fluorescent protein (GFP) from the jellyfish species Aequora victoria . Compared to native GFP, sGFP shows stronger fluorescent and has a shifted excitation wavelength. We demonstrated the potential of this variant as a reporter gene when transiently co-expressed in N. benthamiana . We used this part in the composite part 35S GFP (S65T) BBa_K788009 .

Go To

(BBa_K3757007) 35S Oak1 (BBa_K3757009) 35S sGFP (S65T) (BBa_K3757010) 35S CtAEP1 (BBa_K3757011) 3in1 Oak1 CtAEP1 sGFP (S65T)

( BBa_K3757007 ) 35S Oak1

This part can be used for the constitutive expression of a grafted cyclotide construct embedded in the Oak1 precursor in plants, C-terminally tagged with a c-myc-tag. After expression, the included tag may be used for affinity purification or detection of the precursor with an anti-c-myc-tag antibody in an immunoassay. It inherits the following basic parts:

CaMV 35S promoter ( BBa_K788000 )
Oak1 precursor ( BBa_K3757001 )
c-myc-tag ( BBa_K3757008 )
35S terminator ( BBa_K1159307 )

Feature map of the composite part 35S Oak1. — Figure: Illustration of the genetic features of our composite part 35S Oak1.

( BBa_K3757010 ) 35S CtAEP1

This part can be used for the expression of the cyclizing asparaginyl endopeptidase CtAEP1 in plants, C-terminally tagged with a HA-tag. After expression, the included tag may be used for affinity purification or detection of the CtAEP1 with an anti-HA-tag antibody in an immunoassay. It inherits the following basic parts:

CaMV 35S promoter ( BBa_K788000 )
CtAEP1 ( BBa_K3757000 )
HA-tag ( BBa_K3183024 )
35S terminator ( BBa_K1159307 )

Feature map of the composite part 35S CtAEP1. — Figure: Illustration of the genetic features of our composite part 35S CtAEP1.

( BBa_K3757009 ) 35S sGFP (S65T)

This part can be used for the expression of the fluorescent protein sGFP (S65T) in plants. It inherits the following basic parts:

CaMV 35S promoter ( BBa_K788000 )
sGFP (S65T) ( BBa_K3669012 )
35S terminator ( BBa_K1159307 )

Feature map of the composite part 35S GFP. — Figure: Illustration of the genetic features of our composite part 35S GFP.

( BBa_K3757011 ) 3in1 Oak1 CtAEP1 sGFP (S65T)

Our 3in1 gene cassette is designed to express cyclic peptides or proteins in plants like N. benthamiana . This is faciliated by the co-expression of three genes: The Oak1 precursor ( BBa_K3669012 ) harbors the peptide or protein to get cyclized. A DNA-sequence encoding an arbitraty peptide can be cloned into this precursor in only one highly efficient Golden Gate cloning step. The success of cloning is thereby monitored by blue-white selection. The second gene encodes the cyclizing asparaginyl endopeptidase CtAEP1 ( BBa_K3757000 ). This enzyme recognizes the Oak1 precursor and catalyzes cyclization of the peptide sequence embedded within it. Finally, a GFP variant sGFP (S65T) ( BBa_K3757011 ) is co-expressed as a reporter gene. This enables an easy expression control of the three genes by fluorescence microscopy. The gene cassette is located in a binary vector between the Agrobacteria left and right border, which allow the tranfer and integration of the whole cassette into plant cells and the plant's genome, respectively. This composite part consists of the following basic and composite parts:

A. tumefaciens LB ( BBa_K233326 )
35S Oak1 ( BBa_K3757007 )
35S CtAEP1 ( BBa_K3757010 )
35S sGFP (S65T) ( BBa_K3757009 )
A. tumefaciens RB ( BBa_K233327 )

Feature map of the composite part 3in1 Oak1 CtAEP1 sGFP (S65T). — Figure: Illustration of the genetic features of our composite part 3in1 Oak1 CtAEP1 sGFP (S65T).

About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.