Revision as of 00:00, 19 October 2021

Parts

Parts overview
Basic parts
Composite parts - Groningen Collection

Parts overview

Achieving the highest alpha amylase activity is the main engineering goal of the team this year. We explored the possibilities of Saccharomyces spp. as a cell factory by testing enzymes from different organisms; Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Aspergillus oryzae.

It is sometimes hard to predict which combination of promoter, secretion signal and gene will lead to better yield. Therefore, we aimed to test as many promoters, secretion signal peptides and genes as possible. For this purpose, we used Golden Gate Assembly and took advantage of its modularity to clone our genes into a vector, creating 60 different combinations of promoters, secretion peptides and genes.

The native sequence of alpha amylase codes for a secretion signal that is used in the native host to secrete the enzyme (as these enzymes act extracellularly). To increase the possibilities of achieving higher yields of extracellular protein, besides testing for the native secretion peptide, we isolate the CDS (coding sequence) of alpha amylase and add a different secretion peptide that has been proven to work on yeast^[1]. To know more about the genetic design approach go to the engineering page.

Basic parts

The sequences of alpha amylase from 4 different organisms were codon optimized for the chassis. The basic parts were designed from the native sequence and synthesized de novo. The basic parts can be found in Table 1.

Basic parts designed by iGEM Groningen 2021.
Name	Type	Description	Designer	Length
BBa_K3748000	Coding	Alpha amylase from Bacillus amyloliquefaciens.	iGEM Groningen	1545
BB_K3748001	Coding	Alpha amylase from Bacillus licheniformis.	iGEM Groningen	1539
BB_K3748002	Coding	Alpha amylase from Bacillus subtilis.	iGEM Groningen	1980
BB_K3748003	Coding	Alpha amylase from Aspergillus Oryzae.	iGEM Groningen	1500
BBa_K3748004	Coding	Secreted Alpha amylase from Bacillus amyloliquefaciens.	iGEM Groningen	1545
BB_K3748005	Coding	Secreted Alpha amylase from Bacillus licheniformis.	iGEM Groningen	1539
BB_K3748006	Coding	Secreted Alpha amylase from Bacillus subtilis.	iGEM Groningen	1980
BB_K3748007	Coding	Secreted Alpha amylase from Aspergillus Oryzae.	iGEM Groningen	1500

Composite parts - Groningen Collection

A library of constructs was created (Table 2) in which we included different elements (go to Engineering page) The strategy followed to fuse the different parts can be seen in Figure 1.

Fusion sites used in the MoClo standard for Golden Gate assembly to create the composite parts.

Promoters, secretion signals and terminators are taken from previous characterized genetic parts^[1],[2]. 10 promoters were chosen among the 20 promoters described in Lee ME et al. 2015 based on their strength, selecting promoters representing all strengths, from strong to weak. We also decided to include the most characterized secretion signal, as well as a regular terminator, tTDH1. The process to create the set of samples from all the possible combinations achieving an even distribution of all the elements from the different categories can be found in the model page.

Fusion sites used in the MoClo standard for Golden Gate assembly to create the composite parts.
Name	Type	Description	Designer	Length
BB_K3748020	Composite	SP001 - pPGK1, αMF, BS, tTDH1	iGEM Groningen	3082 bp
BB_K3748021	Composite	SP002 - pRNR1, αMFΔ, AO, tTDH1	iGEM Groningen	2565 bp
BB_K3748022	Composite	SP005 - pHHF1, αMF, BS, tTDH1	iGEM Groningen	3083 bp
BB_K3748024	Composite	SP008 - pHHF1, Part3 BS, tTDH1	iGEM Groningen	2933 bp
BB_K3748025	Composite	SP009 - pRNR1, Part3 BS, tTDH1	iGEM Groningen	2933 bp
BB_K3748026	Composite	SP010 - pHHF1, αMF, BS, tTDH1	iGEM Groningen	3083 bp
BB_K3748028	Composite	SP012 - pRNR1, Part3 AO, tTDH1	iGEM Groningen	2451 bp
BB_K3748029	Composite	SP013 - pRAD27, αMF, BS, tTDH1	iGEM Groningen	2678 bp
BB_K3748030	Composite	SP014 - pREV1, αMF, BA, tTDH1	iGEM Groningen	2678 bp
BB_K3748031	Composite	SP015 - pPGK1, αMFΔ, AO, tTDH1	iGEM Groningen	2565 bp
BB_K3748032	Composite	SP016 - pRAD27, Part3 BL, tTDH1	iGEM Groningen	2486 bp
BB_K3748034	Composite	SP018 - pRAD27, αMFΔ, BA, tTDH1	iGEM Groningen	2576 bp
BB_K3748035	Composite	SP019 - pTEF1, Part3 BL, tTDH1	iGEM Groningen	2492 bp
BB_K3748038	Composite	SP022 - pREV1, αMF, BA, tTDH1	iGEM Groningen	2678 bp
BB_K3748039	Composite	SP023 - pRNR2, αMF_no_EAEA, BL, tTDH1	iGEM Groningen	2666 bp
BB_K3748040	Composite	SP024 - pTEF1, αMFΔ, BL, tTDH1	iGEM Groningen	2582 bp
BB_K3748041	Composite	SP025 - pPAB1, Part3 BA, tTDH1	iGEM Groningen	2498 bp
BB_K3748042	Composite	SP026 - pHHF1, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748043	Composite	SP027 - pRNR1, Part3 BS, tTDH1	iGEM Groningen	2933 bp
BB_K3748044	Composite	SP028 - pTEF1, αMF, BA, tTDH1	iGEM Groningen	2678 bp
BB_K3748045	Composite	SP029 - pRPL18B, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748046	Composite	SP030 - pTDH3, αMFΔ, BA, tTDH1	iGEM Groningen	2562 bp
BB_K3748047	Composite	SP031 - pPGK1, αMFΔ, BA, tTDH1	iGEM Groningen	2582 bp
BB_K3748049	Composite	SP033 - pREV1, αMF, BS, tTDH1	iGEM Groningen	3083 bp
BB_K3748050	Composite	SP034 - pTEF1, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748051	Composite	SP035 - pPAB1, αMF_no_EAEA, BL, tTDH1	iGEM Groningen	2666 bp
BB_K3748052	Composite	SP037 - pRPL18B, αMF, BS, tTDH1	iGEM Groningen	3083 bp
BB_K3748053	Composite	SP038 - pRNR2, αMF, BS, tTDH1	iGEM Groningen	3083 bp
BB_K3748054	Composite	SP039 - pTEF1, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748056	Composite	SP041 - pHHF1, Part3 BA, tTDH1	iGEM Groningen	2498 bp
BB_K3748059	Composite	SP044 - pRNR2, αMFΔ, BL, tTDH1	iGEM Groningen	2582 bp
BB_K3748060	Composite	SP045 - pPAB1, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748061	Composite	SP046 - pTDH3, αMFΔ, BL, tTDH1	iGEM Groningen	2562 bp
BB_K3748062	Composite	SP047 - pPGK1, AO, tTDH1	iGEM Groningen	2451 bp
BB_K3748064	Composite	SP049 - pRAD27, Part3 AO, tTDH1	iGEM Groningen	2445 bp
BB_K3748065	Composite	SP050 - pPGK1, αMF_no_EAEA, BA, tTDH1	iGEM Groningen	2666 bp
BB_K3748066	Composite	SP051 - pRNR1, αMF_no_EAEA, BL, tTDH1	iGEM Groningen	2666 bp
BB_K3748067	Composite	SP052 - pPAB1, αMF, BA, tTDH1	iGEM Groningen	2678 bp
BB_K3748068	Composite	SP053 - pHHF1, Part3 AO, tTDH1	iGEM Groningen	2451 bp
BB_K3748069	Composite	SP054 - pRPL18B, αMFΔ, BS, tTDH1	iGEM Groningen	2987 bp
BB_K3748070	Composite	SP055 - pRPL18B, αMF, BL, tTDH1	iGEM Groningen	2678 bp
BB_K3748071	Composite	SP056 - pPAB1, Part3 BL, tTDH1	iGEM Groningen	2492 bp
BB_K3748073	Composite	SP058 - pRNR2, Part3 AO, tTDH1	iGEM Groningen	2451 bp
BB_K3748075	Composite	SP060 - pREV1, Part3 BS, tTDH1	iGEM Groningen	2933 bp
BB_K3748076	Composite	SP061 - pREV1, αMF_no_EAEA, BA, tTDH1	iGEM Groningen	2666 bp
BB_K3748077	Composite	SP062 - pRAD27, αMF, BA, tTDH1	iGEM Groningen	2672 bp
BB_K3748078	Composite	SP063 - pRNR2, αMF, BL, tTDH1	iGEM Groningen	2678 bp

References

Obst, U., Lu, T. K., & Sieber, V. (2017). A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. ACS Synthetic Biology, 6(6), 1016–1025. https://doi.org/10.1021/acssynbio.6b00337
Lee, M. E., Deloache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol., 4, 975–986. https://doi.org/10.1021/sb500366v

[1] Obst, U., Lu, T. K., & Sieber, V. (2017). A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. ACS Synthetic Biology, 6(6), 1016–1025. https://doi.org/10.1021/acssynbio.6b00337

[2] Lee, M. E., Deloache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol., 4, 975–986. https://doi.org/10.1021/sb500366v

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Difference between revisions of "Team:Groningen/Parts"

Revision as of 00:00, 19 October 2021

Parts overview

Basic parts

Composite parts - Groningen Collection

References