Basic

Basic Parts Five Different Coding Sequences Four Tags for Glycosylation, Detection and Purification Ten Regulatory Units for Chlamydomonas reinhardtii Two Coding Sequences for Antibiotic Resistances Three Secretion Signals for Chlamydomonas reinhardtii Five Scars References

Basic Parts

During our lab phase we designed a large part collection. This was only possible because the Modular Cloning System was introduced in this year’s iGEM competition. To offer future iGEM teams the possibility to use C. reinhardtii as their project chassis, we registered 18 universally applicable L0 parts, as well as two L1 resistance cassettes in the Kaiser-collection. All of them are codon optimized for Chlamydomonas reinhardtii usage. Furthermore, we registered a collection specific for our project idea which contains all L0, L1 and L2 parts that were used during the project phase.

Best basic part

As best basic part we offer the sp20HA-tag which is also a component of the Kaiser collection. In combination with secretion tags of the Kaiser collection, this part allows the secretion of glycosylated proteins. Sp20 contains repeats of serine and proline which act as a glycomodule, while the HA-tag allows detection via antibody-staining. The sp20 tag was used in our project to increase the yield of our secreted proteins MHETase and PETase. By offering this basic part we give future iGEM teams the opportunity to integrate a glycomodule into their gene design which may result in higher secretion rates.

Five Different Coding Sequences

The wildtype PETase is a PET-degrading enzyme that was first discovered in the genome of the bacterium Ideonella sakaiensis. The products of the enzymatic degradation of PET by the PETase are MHET and BHET.
The mutated version of the PETase (mut-PETase) is a version with three point mutations that increase the and activity of the enzyme. For a higher efficiency in combination with C. reinhardtii three introns from the RBCS2 protein were introduced in the sequence of the PETase.
MHETase converts MHET into ethylene glycol and terephthalic acid. Due to its large size, a split version of the protein was registered for better synthesis.
Table 1: PhytoBrick coding sequences submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002004	Coding	Wildtype PETase	Dorothée Klein	1488
BBa_K3002005	Coding	Wildtype MHETase part1	Dorothée Klein	1426
BBa_K3002014	Coding	Mut-PETase	Dorothée Klein	1488
BBa_K3002029	Coding	Wildtype MHETase part2	Marlene Schlosser	1368
BBa_K3002037	Coding	Wildtype MHETase	Dorothée Klein	2469

Four Tags for Glycosylation, Detection and Purification:

During the project, we used different tags for detection and purification of the expressed proteins as well as a tag for glycosylation.
The Sp20-tag contains twenty repeats of serine and proline. Proline is first transformed into hydroxyproline and afterwards glycosylated. The glycosylation increases the secretion level of the protein¹.
The HA-tag can be used to detect the expressed proteins with a primary antibody. Detection with the tag was done by using a primary antibody (anti-HA, mouse) and a secondary antibody (anti-mouse, rabbit) conjugated to horseradish peroxidase (HRP) which allows detection by enhanced chemiluminescence.
The hexahistidine tag can be used for purification and detection. Proteins with His-tags can bind to a Nickel NTA column. The eluted samples from the column can be separated by SDS-PAGE and detected by Coomassie-staining.
Table 2:PhytoBrick tags submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002018	Tag	Sp20 8xHis tag	Marlene Schlosser	153
BBa_K3002028	Tag	6xHis tag	Marlene Schlosser	21
BBa_K3002010	Tag	Sp20-HA tag	Dorothée Klein	222
BBa_K3002017	Tag	3xHA tag	Dorothée Klein	102

Ten Regulatory Units for Chlamydomonas reinhardtii

All promoters and terminators are specific for Chlamydomonas reinhardtii.
The PSAD promoter and terminator naturally encode and terminate for the ferredoxin-binding protein in Chlamydomonas reinhardtii².
The Tubulin 2 (Tub2) promoter and terminator encode and terminate for tubulin, a protein of the cytoskeleton which is expressed constitutively.
The pAR promoter is a fusion of the promoters from the proteins HSP70 and RBCS2. HSP70 is a heat shock protein and therefore the AR promoter is slightly heat-inducible³. RBCS2 encodes for the small chain 2 of rubisco and therefore is expressed constitutively.
Table 3:PhytoBrick terminators and regulators submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002002	Terminator	PSAD terminator	Marlene Schlosser	547
BBa_K3002001	Regulatory	PSAD promoter with 5’UTR	Dorothée Klein	814
BBa_K3002036	Regulatory	PSAD promoter	Marlene Schlosser	776
BBa_K3002003	Regulatory	pAR promoter	Marlene Schlosser	471
BBa_K3002027	Regulatory	pAR promoter with 5’UTR	Marlene Schlosser	494
BBa_K3002006	Terminator	RPL23 terminator	Dorothée Klein	712
BBa_K3002011	Regulatory	Tub2 promoter with 5’UTR	Dorothée Klein	317
BBa_K3002034	Regulatory	Tub2 promoter	Marlene Schlosser	198
BBa_K3002012	Terminator	Tub2 terminator	Dorothée Klein	459
BBa_K3002027	Regulatory	PSAD promoter	Marlene Schlosser	804

Two Coding Sequences for Antibiotic Resistances

These parts encode resistance genes against the antibiotics spectinomycin or hygromycin which allow the selection of transformed cells.
Table 4:PhytoBrick coding sequences submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002000	Coding	Spectinomycin resistance	Dorothée Klein	1026
BBa_K3002013	Coding	Hygromycin resistance	Dorothée Klein	999

Three Secretion Signals for Chlamydomonas reinhardtii

CCA is a secretion peptide derived from the carbonic anhydrase 1⁴. It is a zinc containing metalloenzyme and induces a CO2 concentrating mechanism by reversibly converting CO2 to HCO3^-⁵.
The secretion signal ARS is obtained from the arylsulfatase 1. It is essential for the mineralization of sulfate by hydrolyzing sulfate esters under conditions of sulfate deprivation.6
Also, the secretion peptide of the gametic lytic enzyme (GLE), which is a metalloprotease and mediates digestion of the cell wall7, was used.
Table 5: PhytoBrick Protein domains submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002007	Protein_Domain	cCA secretion signal	Dorothée Klein	63
BBa_K3002008	Protein_Domain	GLE secretion signal	Marlene Schlosser	81
BBa_K3002009	Protein_Domain	ARS secretion signal	Marlene Schlosser	69

Five Scars

Dummys are necessary for a L2 ligation. They connect the ends of the L2 construct with the vector. Since there are eight possible positions for a L2 construct, there is a linker for every number of used constructs.
Table 6: PhytoBrick scars submitted by iGEM TU Kaiserslautern 2019.

Name	Type	Description	Designer	Length
BBa_K3002301	scar	MoClo connector A3-B1	Dorothée Klein	4
BBa_K3002302	scar	MoClo connector B1-B2	Marlene Schlosser	4
BBa_K3002303	scar	MoClo connector B2-B3	Dorothée Klein	1
BBa_K3002304	scar	MoClo connector B4-B5	Marlene Schlosser	4
BBa_K3002305	scar	MoClo connector B5-B6	Dorothée Klein	4

References

[1] Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko (2017): High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii. In: Plant biotechnology journal 15 (9), S. 1214–1224. DOI: 10.1111/pbi.12710.
[2] Part:BBa K1547005 - parts.igem.org. Available online at http://parts.igem.org/Part:BBa_K1547005, checked on 10/17/2019.
[3] Schroda, Michael; Blöcker, Dagmar; Beck, Christoph F. (2000): The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. In: The Plant Journal 21 (2), S. 121–131.
[4] Lauersen, Kyle J.; Berger, Hanna; Mussgnug, Jan H.; Kruse, Olaf (2013): Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. In: Journal of biotechnology 167 (2), S. 101–110. DOI: 10.1016/j.jbiotec.2012.10.010.
[5] Moroney, James V.; Ma, Yunbing; Frey, Wesley D.; Fusilier, Katelyn A.; Pham, Trang T.; Simms, Tiffany A. et al. (2011): The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles. In: Photosynthesis research 109 (1-3), S. 133–149. DOI: 10.1007/s11120-011-9635-3.
[6] Hostos, E. L. de; Togasaki, R. K.; Grossman, A. (1988): Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii. In: The Journal of cell biology 106 (1), S. 29–37. DOI: 10.1083/jcb.106.1.29.
[7] Tetsu Kinoshita; Hideya Fukuzawa; Tomoo Shimada; Tatsuaki Saito; Yoshihiro Matsuda (1992): Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii similarity of functional domains to matrix metalloproteases. In: Cell Biology 89, S. 4693–4697.

Team:TU Kaiserslautern/Basic Part