Difference between revisions of "Team:Bielefeld-CeBiTec/Composite Part"

For our metal resource recovery system we build lots of new basic biobricks for accumulation and storage of metal ions, toxicity counteractions and nanoparticle formation. In order to find the best expression ratios, we tried different regulator elements, such as promoters, ribosome binding sites and terminators in combination with our genes. Some of our proteins were also fused to other proteins with flexible or rigid linkers connecting them. On this page we list all these composite parts and their components.Not only our promotor testing device, but also our (LINK) TACE siRNA testing system consists mainly of composite parts. An important part are the target vectors, which transcribe a target mRNA and express a reporter Protein to allow the user to measure and quantify the silencing efficiency of siRNAs. So we constructed two generations of target vectors, with a member of the second generation being our best composite part:

This Composite Part Our new TACE system consists of an expression vector which transcribes siRNAs, and a target vector which transcribes a target mRNA as well as a reporter protein which enables the user to quantify the silencing ability of a tested siRNA.
The first generation of target vectors featured a direct fusion of a target sequence to a Reporter Protein (Blue fluorescent Protein BFP or AmilCP). This assures the shortest reaction time possible to the degradation of the target mRNA, as the destruction of the target mRNA also leads to the degradation of the marker proteins mRNA. However this has a problem, as a non-functional reporter protein can lead to false positives. This led to the development of the second generation of vectors. This Generation also features an inverter as well as additional control mechanisms. The vector is shown in Figure 1:

This innovative system has the potential to replace CRISPR/Cas in the iGEM contest as it offers as easy and open source way to manipulate the metabolism of an organism.
The part features a Golden Gate Assembly (GGA) Cassette between an ATG and an GGGGS Linker. This allows the user to seamlessly insert a target sequence. Short target sequences can be ordered as oligo nucleotides, annealed and inserted via Golden Gate Assembly. Long Targets can be inserted the same way, if the needed overlaps can be added. Alternatively the Part can be amplified using the Primers in Table XXX, and the target sequence can be inserted via Gibson Assembly after adding matching overlaps. Through the start codon upstream of the GGA cassette the LacI inhibitor will still be produced when a target sequence without an own start codon is inserted. However, the user still has to keep the LacI inhibitor in frame with a start codon. The used terminator B0015has a high terminating ability to prevent random transcription of the reporter protein. Because of the pLac promotor, the time in an experiment when the reporter protein shall be expressed can be chosen freely. BFP was chosen as a reporter protein because it has a low excitation wavelength at 399 nm and emission wavelength at about 450 nm. This recommends the BFP for a FRET system, making further measurements possible.

Table 1: All composite Parts submitted by iGEM Bielefeld 2018.

Identifier	Components	Description	Designer	Length
BBa_K2638003	BBa_K525998, BBa_K2638001	T7 + RBS + CopC	Erika Schneider	425
BBa_K2638004	BBa_K525998, BBa_K2638002	T7 + RBS + CopD	Erika Schneider	950
BBa_K2638005	BBa_I0500, BBa_B0030, BBa_K2638001	T7 + RBS + CopC	Erika Schneider	XXX
BBa_K2638006	BBa_I0500, BBa_B0030, BBa_K2638002	T7 + RBS + CopD	Erika Schneider	XXX
BBa_K2638109	BBa_R0040, BBa_K1460002	PTetR + CRS5	Johannes Ruhnau	1032
BBa_K2638110	BBa_J61101, BBa_K2638103, BBa_J61101, BBa_K2638150	PTetR + gshA + gshB + Phyto	Johannes Ruhnau	2450
BBa_K2638112	BBa_I0500, BBa_B0034, BBa_K2638121, BBa_B0034, BBa_K2638103, BBa_B0034, BBa_K2638120	PTetR + gshA + gshB + GSR	Johannes Ruhnau	4144
BBa_K2638113	BBa_I0500, BBa_B0034, BBa_K2638100	PTetR + ahpC + ahpF	Vanessa Krämer	1800
BBa_K2638114	BBa_R0040, BBa_K554003, BBa_K1104200	PTetR + SoxR + RBS + OxyR	Johannes Ruhnau	1489
BBa_K2638117	BBa_R0040, BBa_J61101, BBa_K2638106	PTetR + RBS + sodA + KatE	Johannes Ruhnau	701
BBa_K2638118	BBa_R0040, BBa_J61101, BBa_K2638106, BBa_J61101, BBa_K2638105	PTetR + RBS + sodA + KatG	Johannes Ruhnau	2989
BBa_K2638201	BBa_K525998, BBa_K2638200	OprC (TonB dependent copper transport porin, BBa_K2638200) with T7 promotor and RBS (BBa_K525998)	Jakob Zubek	2165
BBa_K2638204	BBa_I0500, BBa_B0030, BBa_K2638200	OprC (TonB dependent copper transport porin, BBa_K2638200) with T7 promotor and RBS (BBa_K525998)	Jakob Zubek	2165
BBa_K2638400	BBa_K2638500	Combination of BBa_K2638500 + BBa_K2638560	Levin Joe Klages	889
BBa_K2638401	BBa_K2638502, BBa_K2638426	13	Levin Joe Klages	889
BBa_K2638402	BBa_K2638503, BBa_K2638426	14	Levin Joe Klages	889
BBa_K2638403	BBa_K2638504, BBa_K2638426	15	Levin Joe Klages	889
BBa_K2638404	BBa_K2638506, BBa_K2638426	17	Levin Joe Klages	889
BBa_K2638405	BBa_K2638507, BBa_K2638426	18	Levin Joe Klages	889
BBa_K2638406	BBa_K2638509, BBa_K2638426	110	Levin Joe Klages	889
BBa_K2638407	BBa_K2638510, BBa_K2638426	111	Levin Joe Klages	889
BBa_K2638408	BBa_K2638511, BBa_K2638426	112	Levin Joe Klages	889
BBa_K2638409	BBa_K2638517, BBa_K2638426	118	Levin Joe Klages	903
BBa_K2638410	BBa_K2638520, BBa_K2638426	21	Levin Joe Klages	890
BBa_K2638411	BBa_K26358522, BBa_K2638426	23	Levin Joe Klages	890
BBa_K2638412	BBa_K2638525, BBa_K2638426	26	Levin Joe Klages	890
BBa_K2638413	BBa_K2638526, BBa_K2638426	27	Levin Joe Klages	890
BBa_K2638414	BBa_K2638528, BBa_K2638426	29	Levin Joe Klages	890
BBa_K2638415	BBa_K2638531, BBa_K2638426	212	Levin Joe Klages	890
BBa_K2638416	BBa_K2638532, BBa_K2638426	213	Levin Joe Klages	889
BBa_K2638417	BBa_K2638534, BBa_K2638426	215	Levin Joe Klages	900
BBa_K2638418	BBa_K2638537, BBa_K2638426	218	Levin Joe Klages	904
BBa_K2638419	BBa_K2638542, BBa_K2638426	33	Levin Joe Klages	889
BBa_K2638420	BBa_K2638545, BBa_K2638426	36	Levin Joe Klages	889
BBa_K2638421	BBa_K2638548, BBa_K2638426	39	Levin Joe Klages	889
BBa_K2638422	BBa_K2638551, BBa2638426	312	Levin Joe Klages	889
BBa_K2638423	BBa_K2638554, BBa_K2638426	315	Levin Joe Klages	899
BBa_K2638424	BBa_K26358556, BBa_2638426	317	Levin Joe Klages	909
BBa_K2638425	BBa_K2638557, BBa_K2638426	318	Levin Joe Klages	903
BBa_K2638703	BBa_K2638716, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100	siRNA Target Vector 2 with BFP and GGGGS Linker	Antonin Lenzen	2787
BBa_K2638704	BBa_K2638717, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100	siRNA Target Vector 2 with BFP and GGGGS Linker	Antonin Lenzen	2787
BBa_K2638705	BBa_K2638718, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100	siRNA Target Vector 2 with BFP and cMyc Linker	Antonin Lenzen	2787
BBa_K2638706	BBa_K2638719, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100	siRNA Target Vector 2 with BFP and XP Linker	Antonin Lenzen	2787
BBa_K2638991		araBAD and RBS and Mutated Human Ferritin Heavy Chain (without Stop)	Vanessa Krämer	1210
BBa_K2638997		araBAD and RBS and Human Ferritin Heavy Chain	Vanessa Krämer	552
BBa_K2638998		araBAD and RBS and Mutated Human Ferritin Heavy Chain	Vanessa Krämer