Parts

| NDSU - iGEM 2022

Parts Summary


Our team this year focused on producing a natural occurring dye. We found the chromoproteins from the 2013 Uppsala iGEM team and decided to pursue chromoproteins as an alternative to the environmentally damaging azo dyes.

From last year’s project, the team was already familiar with the CIDAR MoClo Kit and its many options for building tailor made expression vectors. These essential parts like the constitutive promoters, ribosome binding sites, terminators, and backbones were combined with sequences we designed and synthesized to construct dye producing E. coli.

Listed below are a number of gene fragments ((6)-(20)) that we adapted and combined from existing iGEM parts to fit the MoClo Golden Gate cloning scheme. We designed these sequences with the meffRed chromoprotein and two different cellulose binding domains in two orientations. Our constructs with meffRed later proved to show only faint color so we transitioned to the mScarlet fluorescent protein which is much brighter than meffRed even under normal light. We also adapted an arabinose induced promoter to fit our cloning scheme. Parts were codon optimized for E. coli and designed to be Golden Gate compatible with Bsa1 sites for cloning into a circular plasmids for dye production as well as Bpi1 sites for cloning the parts into the pOGG006 backbone for long-term storage in a bacterial stock.

Our project after designing the gene fragments involved cloning these parts together and characterizing the plasmid products as expression vectors for cotton fabric dyes. The rest of the parts listed in the table below are those expression vectors. We went through many unsuccessful combinations and iterations before finding a construct that produced a lot of bright dye while still binding to cellulose (cotton). pGEC046 (BBa_K4258046) is the culmination of our work this year.

Parts Table


Name Name on Tube Description Designers Length
N/A (Not functional) (1)His-meffRed-Linker-CBDfimi Same sequence as (11) except it lacked additional nucleotides that help the enzyme attach and cut. Jake Schumacher N/A
N/A (Not functional) (2)His-meffRed-Linker-CBDclos Same sequence as (12) except it lacked addtional nucleotides that help the enzyme attach and cut. Jake Schumacher N/A
N/A (Not functional) (3)CBDfimi-Linker-meffRed-His Same sequence as (13) except it lacked additional nucleotides that help the enzyme attach and cut. Jake Schumacher N/A
N/A (Not functional) (4)CBDclos-Linker-meffRed-His Same sequence as (14) except it lacked additional nucleotides that help the enzyme attach and cut. Jake Schumacher N/A
N/A (Not functional) (5)meffRed-His_NegControl Same sequence as (15) except it lacked additional nucleotides that help the enzyme attach and cut. Jake Schumacher N/A
BBa_K4258006 (6)araCrepressor+pBAD+RBS Bsa1 sites for Golden Gate cloning as a AC module. araC+pBAD+RBS from BBa_K2457000. Jake Schumacher 1317 bp
BBa_K4258011 (11)His-meffRed-Linker-CBDfimi Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. 6x Histidine tag + MeffRed from BBa_K1033920 + 10x GS Linker + Cellulose Binding Domain from Cellulomonas fimi (BBa_K863101). A coding sequence that will express meffRed (very feint color) with a cellulose binding domain. Jake Schumacher 1129 bp
BBa_K4258012 (12)His-meffRed-Linker-CBDclos Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. 6x Histidine tag + MeffRed from BBa_K1033920 + 10x GS Linker + Cellulose Binding Domain from Clostridium cellulovorans (BBa_K863111). A coding sequence that will express meffRed (very feint color) with a cellulose binding domain. Jake Schumacher 1102 bp
BBa_K4258013 (13)CBDfimi-Linker-meffRed-His Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. Cellulose Binding Domain from Cellulomonas fimi (BBa_K863101) + 10x GS Linker + MeffRed from BBa_K1033920 + 6x Histidine tag. A coding sequence that will express meffRed (very feint color) with a cellulose binding domain. Jake Schumacher 1129 bp
BBa_K4258014 (14)CBDclos-Linker-meffRed-His Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. Cellulose Binding Domain from Clostridium cellulovorans (BBa_K863111) + 10x GS Linker + MeffRed from BBa_K1033920 + 6x Histidine tag. A coding sequence that will express meffRed (very feint color) with a cellulose binding domain. Jake Schumacher 1102 bp
BBa_K4258015 (15)meffRed-His_NegControl Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. MeffRed from BBa_K1033920 + 6x Histidine tag. Used as a negative control against parts BBa_K4258011 through BBa_K4258014 which have cellulose binding domains. A coding sequence that will express meffRed (very feint color) with a cellulose binding domain. Jake Schumacher 772 bp
BBa_K4258016 (16)His-mScarlet-Linker-CBDfimi Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. 6x Histidine tag + mScarlet from Addgene Plasmid #121003 + 10x GS Linker + Cellulose Binding Domain from Cellulomonas fimi (BBa_K863101). A coding sequence that will express m-Scarlet-I (bright pink) with a cellulose binding domain. Jake Schumacher 1123 bp
BBa_K4258017 (17)His-mScarlet-Linker-CBDclos Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. 6x Histidine tag + mScarlet from Addgene Plasmid #121003 + 10x GS Linker + Cellulose Binding Domain from Clostridium cellulovorans (BBa_K863111). A coding sequence that will express m-Scarlet-I (bright pink) with a cellulose binding domain. Jake Schumacher 1096 bp
BBa_K4258018 (18)CBDfimi-Linker-mScarlet-His Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. Cellulose Binding Domain from Cellulomonas fimi (BBa_K863101) + 10x GS Linker + mScarlet from Addgene Plasmid #121003 + 6x Histidine tag. A coding sequence that will express m-Scarlet-I (bright pink) with a cellulose binding domain. Jake Schumacher 1129 bp
BBa_K4258019 (19)CBDclos-Linker-mScarlet-His Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. Cellulose Binding Domain from Clostridium cellulovorans (BBa_K863111) + 10x GS Linker + mScarlet from Addgene Plasmid #121003 + 6x Histidine tag. A coding sequence that will express m-Scarlet-I (bright pink) with a cellulose binding domain. Jake Schumacher 1096 bp
BBa_K4258020 (20)mScarlet-His_NegControl Bsa1 sites for Golden Gate cloning as a CD module and Bpi1 sites for cloning into pOGG006 to immortalize the part as a plasmid. mScarlet from Addgene Plasmid #121003 + 6x Histidine tag. Used as a negative control against parts BBa_K4258016 through BBa_K4258019 which have cellulose binding domains. A coding sequence that will express m-Scarlet-I (bright pink) with a cellulose binding domain. Jake Schumacher 766 bp
N/A (Not functional) pGEC021 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC023 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC025 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC026 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC028 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC031 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
N/A (Not functional) pGEC035 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
BBa_K4258036 pGEC036 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + c99 mScarlet-I from CIDAR MoClo Extension Kit + B0015 Terminator (BBa_K4258016) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4363 bp
BBa_K4258037 pGEC037 The Golden Gate product of the following parts: J23102 Promoter (BBa_J23102) from the MoClo CIDAR Kit + BCD2 RBS (BBa_K1114107) from the MoClo CIDAR Kit + c99 mScarlet-I from CIDAR MoClo Extension Kit + B0015 Terminator (BBa_K4258016) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 3191 bp
BBa_K4258046 pGEC046 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + Synthesized CDS #16 + B0015 Terminator (BBa_K4258016) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4738 bp
BBa_K4258047 pGEC047 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + Synthesized CDS #17 + B0015 Terminator (BBa_K4258017) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4711 bp
BBa_K4258048 pGEC048 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + Synthesized CDS #13 + B0015 Terminator (BBa_K4258018) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4744 bp
BBa_K4258049 pGEC049 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + Synthesized CDS #19 + B0015 Terminator (BBa_K4258019) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4711 bp
BBa_K4258050 pGEC050 The Golden Gate product of the following parts: AraC Inducible Promoter (BBa_K4258006) + Synthesized CDS #20 + B0015 Terminator (BBa_K4258020) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 4381 bp
N/A (Not functional) pGEC055 No pink colonies or cultures were ever observed. Either the sequence is faulty or there was an error in our labwork. Jake Schumacher N/A
BBa_K4258060 pGEC060 The Golden Gate product of the following parts: J23102 Promoter (BBa_J23102) from the MoClo CIDAR Kit + BCD2 RBS (BBa_K1114107) from the MoClo CIDAR Kit + Synthesized CDS #20 + B0015 Terminator (BBa_K4258016) from the CIDAR MoClo Kit + DVK_AE Backbone from the CIDAR MoClo Kit. Jake Schumacher 3209 bp
N/A (Not functional) pGEC062 No blue colonies or cultures were ever observed. The sequence was incorrectly designed. Jake Schumacher N/A