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Basic Parts

Registered Number Name Description Sequence
BBa_K5149000 Codon Optimized CbAgo CbAgo is a part of the Argonaute protein family. They are known for gene silencing and protection against exogenous DNA/RNA. In our project, we use CbAgo as a method for genome manipulation. By providing a plasmid with regions homologous to the target genome and CbAgo, DNA guides are produced by CbAgo and taken in to form full complexes. These complexes are then to cut every part of the genome where the DNA guide binds. CbAgo is a mesophile meaning it functions between 20-45 degrees Celsius compared to most thermophile Argonaute proteins. ATGAATAACTTGACTTTTGAGGCTTTTGAGGGTATTGGTCAACTGAATGAACTGAACTTTTATAAATACCGGCTCATTGGTAAAGGGCAGATAGATAATGTTCATCAGGCTATCTGGTCTGTTAAATATAAATTACAGGCTAACAACTTCTTTAAACCTGTTTTTGTTAAAGGTGAAATTCTGTATTCTTTAGATGAATTAAAGGTTATTCCTGAGTTTGAGAATGTTGAGGTTATTCTTGACGGTAACATTATTTTGTCTATCTCTGAAAATACTGATATTTATAAAGACGTTATTGTTTTTTATATCAATAATGCTTTGAAGAACATTAAAGACATTACGAATTACCGTAAATACATTACTAAGAATACTGATGAAATTATCTGTAAGTCTATCTTGACTACTAACTTGAAGTATCAATATATGAAGAGTGAGAAAGGTTTTAAATTACAACGTAAATTCAAAATATCTCCTGTTGTATTTAGAAACGGTAAGGTTATTTTGTATTTAAACTGTTCTTCTGATTTTTCTACTGATAAAAGTATTTATGAGATGTTGAATGACGGTTTAGGTGTTGTTGGTTTACAGGTTAAGAATAAATGGACTAACGCTAATGGAAATATCTTTATTGAAAAAGTTCTTGATAACACTATTTCTGATCCTGGAACTTCTGGTAAATTAGGGCAGTCTCTCATTGACTACTACATTAACGGTAACCAAAAATATAGAGTTGAGAAATTCACTGATGAAGATAAAAACGCTAAGGTTATTCAGGCAAAAATAAAGAATAAAACCTATAACTATATTCCTCAGGCTTTAACTCCTGTTATAACTAGAGAGTATTTATCTCATACTGATAAAAAGTTCAGCAAACAGATTGAGAACGTTATAAAAATGGACATGAATTATCGGTATCAAACATTAAAGTCTTTTGTTGAAGATATAGGTGTTATAAAAGAATTAAATAACCTACACTTCAAGAACCAATACTATACTAATTTTGACTTTATGGGTTTTGAATCTGGTGTTTTAGAAGAACCTGTTTTAATGGGAGCTAACGGGAAAATAAAAGACAAGAAACAGATATTCATTAATGGTTTCTTTAAGAACCCTAAAGAGAACGTTAAGTTTGGGGTTTTATATCCTGAAGGATGTATGGAGAATGCTCAGTCTATAGCCCGTTCTATCTTAGATTTTGCCACTGCTGGTAAATATAACAAACAGGAAAATAAATATATTTCTAAAAACCTCATGAATATAGGTTTTAAACCCTCTGAATGTATTTTTGAATCCTATAAATTAGGAGACATTACTGAATATAAGGCTACTGCCCGTAAATTAAAAGAACATGAAAAAGTTGGTTTTGTTATTGCTGTTATTCCTGATATGAATGAGTTAGAAGTTGAAAATCCTTACAATCCTTTTAAAAAGGTTTGGGCTAAATTAAACATTCCCTCACAAATGATCACATTAAAAACTACTGAGAAATTCAAAAATATAGTTGATAAAAGCGGTTTATATTATTTACACAATATAGCTTTAAATATCCTGGGTAAGATAGGTGGTATTCCTTGGATCATTAAAGATATGCCTGGAAATATAGACTGTTTTATTGGTTTAGATGTTGGTACTAGAGAAAAAGGTATTCATTTTCCTGCTTGTTCTGTTTTGTTTGATAAATACGGTAAATTGATAAATTACTATAAACCTACCATTCCTCAGTCTGGTGAGAAGATAGCCGAAACCATTCTTCAAGAGATTTTTGATAATGTTTTAATTTCCTATAAAGAAGAGAATGGTGAATACCCTAAAAATATAGTTATTCACCGGGACGGTTTTTCTAGGGAAAATATAGATTGGTATAAAGAGTATTTTGATAAAAAGGGAATTAAATTCAACATTATTGAGGTTAAGAAAAACATTCCTGTTAAGATAGCTAAAGTTGTTGGTAGTAATATCTGTAATCCTATCAAAGGTAGTTATGTTTTGAAAAACGACAAAGCCTTTATTGTTACTACTGATATAAAAGATGGTGTTGCTTCCCCTAATCCTTTAAAAATAGAGAAAACCTATGGTGATGTTGAGATGAAGAGTATTCTTGAACAGATATATTCTTTATCCCAAATTCATGTTGGTTCTACTAAGTCTTTACGTTTACCTATAACTACTGGATATGCTGATAAAATCTGTAAGGCTATTGAATATATTCCTCAAGGTGTTGTTGATAACCGTTTATTTTTTTTATAA
BBa_K5149001 Up Homology Arm to AquI Locus The Up Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region. To use the Up Homology Arm to AquI Locus, you need to pair it with the Down Homology Arm to AquI Locus. In addition, you need to include some gene in between the Arms, for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. By doing this, you now have given your organism a template for homologous recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination allowing for GFP to be inserted into the genome. This is how we used the Up Homology Arm in our project. CCGTTATCCCATCTGCAATATCCATAAAAGGCGATCGCCTACCCTTCGAGAGACTTGATCCACTCAAGGTAGTATTGTCCTGATCCCTAAGAGATAATCTTTAGGTGAGCTGTACAATAACACAAAATTCATAATGGAAAAAAAACTGATAAGCCTATTTGCCGGAGCTGGTGGCATGGACATCGGCTTTCACGCTGCTGGCTTCAGTACGGCGGTAGCTGTGGAACAAGATCCGTCTTGCTGCAATACTCTAAGGCTTAATATGCCGGACACTCCAGTCATTGAAGGCGACATCACCTCAATTACAACACAGGCTATCTTAGAAGCGGCAAAGGTCAATCCTCTTGAAATTGACCTAGTTATTGGTGGCCCCCCTTGCCAAAGTTTCAGCCTAGCAGGTAGACGTATGGGAATGGATGATCCGAGAGGAATGCTGGTACTTGAGTTTCTACGTGTAGTCAGGGAGGCATTGCCAAAGTGTTTTGTTATGGAGAATGTCAAAGGAATGATCAATTGGTCAAAGGGAAAGGCTCTTGAGGCTATCATGACAGAAGCATCGCAGCCTATAAAATACGCTGGAAAAGAATATAAATACGCTGTTTCGTATCATGTCCTAAATGCTGCTGATTTTGGTGTTCCGCAATTTAGAGAAAGAGTATTCATCGTAGGTAATCGTTTGGGCAAAACATTCCAATTTCCTGAACCAACTCATGGGCCTAGCAACCAAGCGAGACAGATAGATCTTTTTGGCAAGCAGCTAAAACCTTACAAAACTGTTCAAGATGCAATTAGCACTCTCCCCCCTGCAACCCCTCCTTCAGCGATGGCACTAAGAGTTTCGCAGACTATAAAAGATAGGATAAAGAATCATGGATATTAAAAACGTTCATATCAAAAATCACGAACAAACAGCTCATGCACCTTCCACTCTAGAAAAAATTCGTAAAGTCAAACAAGGGGGTAAACTCTCAGAACAGACAAAGACATTTGGTTCAACCTACCGCAGGTTAGATCCGAACCAGCCATCTCCTACAGTGACCCGTAGTGGTTATCGAGATTTTATTCATCCTTTTGAAGATCGAATGCTCACAGTTCGTGAACTGGCTTGTTTGCAAACCTTTCCCCTTGATTGGGAGTTTACCGGAACTCGACTTGATTCTTATAGTAGTAAACGTAAAGTGGCGATGACTCAGTTTGGACAAGTGGGTAATGCAGTACCACCGTTACTTGCTGAAGCTGTTGCTAAAGCGGTTAGCGAACAGCTTCTGGATGTCATT
BBa_K5149002 Down Homology Arm to AquI Locus The Down Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region. To use the Down Homology Arm to AquI Locus, you need to pair it with the Up Homology Arm to AquI Locus. In addition, you need to include some gene in between the Arms, for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. By doing this, you now have given your organism a template for homologous recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination allowing for GFP to be inserted into the genome. This is how we used the Down Homology Arm in our project. CAAAATCAAACCCGATCGCCTCTCTATTTTGATAAATCTATGTCTACTCCCTCTGTTACCCCTGTAGAATCTAGCACCCTAATCAAAACCCCTGAACTGCTGGCTCCAGCGGGAAATTGGGACTGTGCGATCGCCGCCGTAGAAAATGGAGCCGATGCGATTTATTTTGGGCTGGATAAATTTAATGCCCGGATGCGATCGCAAAACTTTGTCGAGTCAGATTTGCCGGAGTTAATGGCATACTTACATCGGCGTGGCGTGAAGGGCTATGTGACGTTAAATACGCTGATTTTCACCTCGGAATTGGCGGCAGTCGAACAATATTTGCGGTCGATTATTGCGGCAGGAGTCGATGCGGCGATTGTCCAGGATGTGGGGCTATGTCAGTTAATTCGGCAATTGTCCCCCGATTTTCCGATCCATGGTTCCACCCAGATGACCGTCACCAGCGCCGCAGGGGTCGAGTTCGCGCAAAACTTGGGTTGTGATTTGGTGGTATTGGCGCGGGAATGTTCGATCAAGGAAATCAATAAAATCCAGCAGGAATTGGGTCAACAAAAGATCTCGATGCCGCTGGAGGTGTTTGTTCATGGAGCGTTGTGTGTCGCCTATTCCGGGCAATGTTTAACCAGTGAATCCCTCGGTGGACGGTCGGCCAATCGCGGGGAATGCGCCCAGGCTTGCCGGATGCCCTACGAAATGATTGTCGATGGTAAGCCATTTGATCTGGGTGACAGACGTTACCTACTAAGCCCCCAAGATTTGGCGGGATTACCTGTTTTGCCGGACTTAATTAAAGCGGGCGTCGTGTCGCTAAAAATCGAAGGACGCTTAAAACAACCGGAATATGTGGCCAGTGTCACCCAAGTGTATCGCCAGGCCATTGATCGGGCGGTGCAGGGCATTGAGCAAGAGGTTTCAGACCAGGAAAAATATCAATTGGAAATGGCCTTTTCGCGTGGGTTGTACACGGGTTGGCTTGACGGCATCGATAATCAAAGCCTTGTCCATGGTCGCTTTGGCAAGAAAAG
BBa_K5149003 Chi Site Chi Sites are repeated DNA sequences stopping recognition sites for RecBCD complexes. For our project, Chi Sites regulates the complete digestion of the dsDNA region we are attempting to edit. By implementing Chi Sites in between each of the 400bp regions of AquI Locus, we can test the efficiency of CbAgo taking in a DNA Guide to form a full complex. To test this, the speed at which the AquI Locus is cut will determine whether or not CbAgo will depend on Chi Sites for more efficient guide acquisition. DNA acquisition for CbAgo is only possible with RecBCD or a RecBCD-like system. GCGATCGC
BBa_K5149004 AquI-1 - 1-400 bp region of AquI Locus The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 1-400 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides. AACAATTTCAGTGACCTTGTGACGACAAAAGATGCTCGCAGAAGTGGTTTTCTTGAATATGCTCTTCGTCGCAATAAAGAGTCTATTCCATTTATCGATAAGGCGAAAGCTCTACAAGTATCTCTTCAGAAGAATACAAAGTGTGCAGAGGATATTCTCAAACTCTATGACATCCGAGAAACACTCATTGAGGCAGCGGGTGTATCTGTCAAGGCGAAGGCTCACCTCGATGACATGGACAAAAATGAAATTCTTGCAGGCTTTGTTAAGGAGGTTCTTGCACCTTGTGGCAAAAAGTATATCGATGAGATTGTCTATCGTTACTTGCTCACACTTG
BBa_K5149005 AquI-2 - 401-800 bp region of AquI Locus The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 401-800 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides. TCGTTACTTGCTCACACTTGGAGATGCTCTTGGTGGTAAAATGCGCAACATTGTTGGTTCTATCGCAGAAGAGAAATTCGTCAGATTTATCGTTGCCCAGTTACAGGTGCATGATATTGAATTTGAACTATTTCCAAAACGTTCGCAATGGATTTCAAACAAAAACTATAGAGCTGAGATGGCTGCACGAACGAAAGCCTTAAGATGGAAAAATAAAACAACTTATCGATCTCTAATTTTCAACATTAATGTTCCACAGGTCAAAAAGAATATTGATATCGTGCTGCTAAATGACAAGATTGAAGGAGTAACCTCGTCTATTTTGGCGCT
BBa_K5149006 AquI-1 - 801-1200 bp region of AquI Locus The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 801-1200 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides. GGAGTAACCTCGTCTATTTTGGCTAATATCCTTAACGATAAGAATAAGTATCTTGCCATCGGTGAGTTAAAGGGCGGAATTGATCCAGCCGGGGCAGATGAACATTGGAAAACGGCAAATACAGCTCTTAGTAGAGTAAGAGATTCTTTCAAGAACAAAATCCACTTATTTTTCATTGGGGCTGCGATAGAAACCAGTATGTCGGCAGAGATTTACGAACAATGCCAGACAGGTGAACTTAGTAACGCAGCAAATCTCACTGTTGATAATCAATTATCTGCATTGTGTGAGTGGCTAATAACGCAATAACTTTACAAAATCAAACCCGATCGC
BBa_K5149007 Codon Optimized Cpf1 Cpf1 is a type 2 Cas protein complex, also known as Cas12a. Using single stranded guide RNA, this complex is able to induce double breaks in the genome of the host to induce homologous recombination. The guide RNA directs the complex to the desired site through a PAM sequence, allowing high specificity when paired with the proper sgRNA cassette. Cpf1 has been shown to have low toxicity in other strains of cyanobacteria compared to other type 2 Cas systems. This version of Cpf1 has been optimized by our BLACKBIRD software to ensure the lack of type IIS recognition site and to represent the native codon bias in UTEX 3154. ATGTCTATATATCAAGAGTTTGTGAATAAATATAGTCTGAGTAAAACTCTACGCTTCGAGTTAATTCCACAGGGTAAAACACTTGAGAACATAAAGGCTAGAGGTTTAATTCTTGATGATGAGAAGAGAGCTAAAGACTACAAGAAGGCTAAGCAAATCATTGATAAATATCATCAGTTCTTTATAGAGGAGATACTAAGTTCTGTTTGTATTTCTGAGGATTTACTACAAAACTACTCTGATGTTTACTTTAAACTGAAGAAGAGTGATGATGATAACCTACAGAAAGACTTCAAATCCGCAAAAGATACCATAAAGAAACAAATAAGTGAATATATAAAAGACAGTGAGAAATTCAAGAATTTGTTTAACCAAAACCTCATTGACGCTAAGAAAGGGCAGGAGTCTGATCTAATTCTGTGGCTAAAGCAGTCTAAGGATAACGGTATTGAATTATTTAAGGCTAATTCTGATATAACAGATATAGATGAGGCGTTAGAGATAATCAAGTCTTTTAAAGGCTGGACAACATACTTTAAGGGGTTTCATGAGAATAGGAAGAATGTTTATTCCAGCAACGACATACCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTCCTGGAGAATAAAGCAAAGTATGAGAGTTTGAAAGACAAAGCTCCTGAGGCTATAAATTATGAACAAATTAAGAAGGATTTAGCCGAAGAGCTAACCTTTGATATTGACTACAAGACCTCTGAGGTTAACCAAAGAGTGTTCTCACTTGATGAGGTATTCGAGATAGCTAACTTTAACAACTATCTAAACCAGAGCGGAATTACTAAATTCAACACTATTATTGGTGGTAAATTCGTTAATGGTGAGAATACAAAGCGTAAAGGTATCAATGAATATATAAACCTATACTCACAGCAGATAAACGACAAAACACTCAAGAAATATAAGATGAGTGTCTTATTTAAGCAAATTTTAAGTGATACAGAGTCTAAGTCTTTTGTTATTGATAAACTGGAAGATGACTCTGATGTAGTTACAACGATGCAGTCCTTCTATGAGCAGATAGCCGCTTTTAAAACAGTAGAAGAGAAGAGCATCAAAGAAACACTATCTTTATTGTTTGATGATTTAAAAGCTCAGAAACTGGATTTGTCGAAGATATACTTTAAGAACGATAAATCTCTTACTGATCTAAGTCAGCAGGTATTCGATGACTATAGTGTTATTGGTACAGCGGTACTGGAATATATAACTCAACAAATAGCACCTAAGAACCTTGATAACCCTAGTAAGAAAGAGCAGGAACTGATAGCCAAGAAAACTGAGAAAGCAAAGTACCTATCCCTAGAAACTATAAAGTTGGCGTTGGAAGAGTTTAACAAACATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCCAACTTTGCCGCTATACCGATGATCTTTGATGAAATAGCTCAGAACAAAGATAATTTGGCACAAATATCTATAAAGTATCAAAACCAAGGTAAGAAAGACCTACTTCAGGCTTCTGCGGAAGATGACGTTAAGGCAATCAAGGATCTGTTAGATCAAACCAATAATTTGTTACATAAACTAAAGATATTTCACATCAGTCAGTCTGAAGATAAAGCAAACATCTTAGACAAAGATGAGCATTTCTATCTGGTCTTTGAGGAGTGTTACTTTGAGCTAGCCAATATAGTGCCTCTTTATAACAAAATAAGAAACTATATAACTCAGAAACCATACAGTGATGAGAAATTCAAGCTCAATTTTGAAAACTCGACGTTGGCTAATGGTTGGGATAAGAATAAAGAGCCCGACAATACAGCCATTCTGTTTATCAAAGATGATAAATACTATTTAGGTGTGATGAATAAGAAGAACAACAAGATCTTTGATGACAAAGCTATCAAGGAAAATAAAGGTGAGGGTTATAAGAAGATAGTTTATAAACTTTTGCCTGGGGCAAACAAAATGTTGCCTAAGGTTTTCTTCTCTGCTAAGTCTATAAAGTTCTATAACCCCAGTGAAGATATATTGAGAATAAGAAACCATTCCACACATACAAAGAATGGTTCACCTCAGAAGGGATATGAGAAGTTCGAGTTTAACATTGAGGATTGCCGAAAATTCATAGACTTTTATAAACAGTCTATCAGTAAACATCCTGAATGGAAGGATTTTGGCTTTAGGTTTTCTGATACTCAAAGATATAACTCTATAGATGAGTTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATAAGTGAGAGCTATATTGATAGTGTAGTTAATCAGGGTAAGTTGTACCTATTCCAGATATATAACAAAGACTTTTCAGCCTACAGCAAAGGACGCCCAAACCTACATACTTTATACTGGAAAGCTCTGTTCGATGAGAGAAACCTTCAAGATGTGGTTTATAAGTTGAATGGTGAGGCAGAGCTTTTCTACCGTAAACAGTCAATACCTAAGAAAATAACTCATCCAGCCAAAGAGGCTATAGCCAATAAGAACAAAGATAATCCTAAGAAGGAGAGTGTGTTCGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAATTCTTCTTCCACTGTCCTATAACAATCAACTTTAAAAGTAGTGGAGCTAATAAGTTCAATGATGAAATTAATTTGTTACTAAAAGAGAAAGCTAATGATGTTCACATACTGAGTATAGATAGAGGTGAGAGACATTTAGCCTACTATACCTTGGTTGACGGTAAGGGCAATATCATCAAACAGGATACATTCAACATCATTGGTAACGACAGAATGAAAACAAACTACCATGATAAATTGGCTGCCATTGAAAAGGACAGGGATTCAGCCCGTAAAGACTGGAAGAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAGATAGCTAAGTTGGTTATAGAGTATAACGCCATTGTGGTATTCGAGGATTTAAATTTTGGCTTTAAAAGAGGGCGTTTCAAGGTAGAGAAACAGGTTTATCAAAAGCTGGAGAAAATGCTCATTGAGAAACTAAACTATCTGGTGTTCAAAGATAATGAGTTTGATAAAACAGGAGGAGTTTTAAGAGCCTATCAACTAACAGCACCGTTTGAGACTTTTAAGAAAATGGGTAAACAAACAGGAATTATCTATTATGTACCCGCTGGTTTTACTTCAAAGATCTGTCCTGTAACTGGTTTTGTAAATCAGTTATACCCCAAGTATGAGAGCGTCAGCAAAAGTCAAGAGTTCTTCTCTAAGTTCGACAAAATCTGTTATAACCTTGATAAAGGCTATTTTGAGTTTAGCTTTGACTATAAGAACTTTGGAGACAAGGCTGCCAAAGGTAAATGGACTATAGCTAGCTTTGGCAGTAGGTTGATTAACTTTAGAAATTCTGATAAGAATCATAATTGGGATACTAGAGAGGTTTACCCAACTAAAGAGCTAGAGAAATTGCTAAAAGATTACTCTATCGAGTATGGTCATGGTGAATGTATCAAAGCCGCTATCTGCGGTGAGTCTGATAAGAAGTTCTTTGCCAAACTAACTTCTGTTCTAAATACTATCTTACAAATGCGTAACTCAAAGACAGGAACTGAGTTAGACTATCTAATTTCCCCAGTAGCGGATGTGAATGGCAACTTCTTTGATAGCCGACAAGCGCCCAAGAATATGCCTCAAGATGCTGATGCCAATGGTGCGTATCATATTGGGCTAAAGGGGCTGATGCTCCTGGGTCGTATCAAGAACAACCAAGAGGGCAAGAAGCTCAATCTGGTGATCAAGAATGAAGAGTATTTTGAATTTGTACAGAACAGGAATAACTAA
BBa_K5149008 Codon Optimized EGFP EGFP is commonly used throughout the world of synthetic biology as a fluorescent market. Specifically in cyanobacteria, GFP works well due to its emission range not overlapping with that of the native fluorescence that comes from phycocyanin. The excitation wavelength of the fluorescent marker is 488nm while the emission wavelength is 507 nm. This version of EGFP has been optimized by our BLACKBIRD software to ensure the lack of type IIS recognition site and to represent the native codon bias in UTEX 3154. ATGGTGAGTAAGGGAGAGGAACTGTTCACAGGGGTAGTGCCTATCCTCGTCGAACTGGACGGAGATGTAAACGGGCATAAGTTCAGCGTTTCCGGTGAGGGTGAAGGTGATGCCACCTACGGCAAACTAACCCTGAAGTTTATCTGTACCACAGGCAAACTGCCCGTGCCTTGGCCTACCCTCGTCACTACCCTAACCTACGGCGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAACAACATGACTTTTTCAAAAGTGCCATGCCCGAAGGCTACGTTCAGGAACGCACTATCTTCTTCAAGGATGACGGCAACTATAAGACTAGAGCCGAGGTGAAGTTCGAGGGTGATACTCTGGTGAACCGCATAGAGTTAAAGGGCATTGACTTTAAAGAGGACGGCAACATCCTGGGGCACAAACTGGAATACAACTACAACAGCCACAACGTCTATATCATGGCAGACAAACAGAAGAACGGCATCAAGGTGAACTTCAAGATACGCCACAACATAGAGGACGGCAGCGTACAGTTAGCCGACCACTATCAGCAGAACACCCCCATCGGTGATGGCCCAGTGCTGCTGCCCGACAACCACTACCTGAGTACCCAGTCTGCCCTGAGCAAAGACCCCAACGAGAAGCGGGATCACATGGTCCTGTTAGAGTTTGTTACCGCAGCAGGAATCACTCTGGGTATGGACGAACTGTATAAATAA
BBa_K5149009 sgRNA Cassette The sgRNA cassette consists of seven unique sgRNAs targeting the AquI locus. The sgRNAs were generated using the sgRNA tool, CasFinder, and were designed to be oligos that were annealed by their complementarity on the sgRNA fragment. TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGCTGATTTAGGCAAAAACGGAATTTCTACTGTTGTAGATGTAAACAATTTCAGTGACTGGCAATTTCTACTGTTGTAGATTAAACAATTTCAGTGACCTTCTAATTTCTACTGTTGTAGATCAGTGACCTTGTGACGACCAATAATTTCTACTGTTGTAGATCTGCGTTACTAAGTTCACTATGAATTTCTACTGTTGTAGATGCTGCGTTACTAAGTTCAAGAAAATTTCTACTGTTGTAGATACCTGTCTGGCATTGTTCGCTGAATTTCTACTGTTGTAGATTTCGTAAATCTCTGCCGAAACGAATTTCTACTGTTGTAGATCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCC
BBa_K5149010 Up Homology Arm to Editing Vector This homology arm allows for recombination between the Editing Strain and the Hybrid Suicide Plasmid to select for integrants with the new antibiotic resistance. To generate this part, flagged primers were designed flanking a 507 bp region on the pC1.509 construct from the Victoria et al (2024) publication. To obtain the part, PCR was carried out to amplify the desired homology arm. ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGCTCGAGTTACCAATGCTTAATCAG
BBa_K5149011 Down Homology Arm to Editing Vector This homology arm allows for recombination between the Editing Strain and the Hybrid Suicide Plasmid to select for integrants with the new antibiotic resistance. To generate this part, flagged primers were designed flanking a 517 bp region on the pC1.509 construct from the Victoria et al (2024) publication. To obtain the part, PCR was carried out to amplify the desired homology arm. TGTGTACCCGGACAAACACCATTAATCAGCAGGAAGGTAAATTCTTCAATATCCTGTTCAACGGTCAACTGTTCGGTCAGCAGGCGATACCAACAAAAACCAAAAATCATATCCAGCAGCAGTTCACGATTGATATCTTTCGGCAGTTCACCATTGCTAATGGCATCTTCAACCAGTTTTTTCGGTATCTCACGACGACGTTCCATAAACTGATCTTTCAGTTGGGTCAGGGTTACAGGGTCCAACTGTGCTTCTGCAATAACACAACGAAATGCTTCACCACAAATGGTTTCACGCCAAACTTTCCACAGATTATGCAGCAGAAAATCCAGATCGGCTTTAAAGCTACCCAAATCCGGAAATTTACGTACCTGTTCGATTTCATTTTCATACACTTCGGCAATCAGTGCTGCTTTGTTGGTCCACCAACGATAAATGGTCGGTTTGCCTGCACCGGCGCGACGTGC
BBa_K5149012 Codon Optimized Gentamicin Codon optimized and STEALTHed Gentamicin Resistance cassette, complete with Level 0 overhangs ready for Level 1 cloning. Gentamicin is an antibiotic that is used for selectivity of our parts. It gives us more flexibility. ATGTTACGCAGCAGCAACGACGTTACGCAGCAAGGTAGCCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATCCGCACATGTAGGCTCGGTCCTGACCAAGTTAAATCCATGAGGGCTGCTCTTGACCTTTTTGGTCGTGAGTTTGGAGACGTAGCCACCTACAGTCAACACCAGCCGGACAGTGATTACTTAGGGAACTTGCTCCGTAGTAAGACATTCATTGCGCTTGCAGCCTTTGACCAAGAAGCGGTTGTGGGTGCTCTGGCGGCTTACGTTTTACCTAGGTTTGAGCAGCCGCGTAGTGAAATCTATATCTATGACTTAGCCGTCTCCGGTGAGCACCGGAGACAGGGCATTGCCACCGCTCTCATCAACCTCCTCAAACATGAGGCTAACGCTCTTGGGGCTTATGTGATTTACGTGCAAGCGGATTACGGTGACGACCCCGCAGTGGCTCTCTATACAAAATTGGGCATACGGGAAGAAGTGATGCACTTTGATATTGACCCAAGTACCGCCACCTAA

Composite Parts

Registered Number Name Description
BBa_K5149100 araBAD pBAD promoter (BBa_I13453), CbAgo, and rrnB T1 Terminator (BBa_B0010) The composite part is used to induce the plasmid expression of CbAgo when transformed into the cell. By doing this, we can study the toxicity of CbAgo based on the amount of Arabinose we provide to our organism. In addition, we can allow our replicative accelerator plasmid to grow in the cell before CbAgo starts to cut and destroy the plasmids.
BBa_K5149101 araBAD pBAD promoter (BBa_I13453), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010) Although Cpf1 has been shown to have a low toxicity level compared to other Cas protein complexes, the introduction of the areBAD promoter allows the user to keep better control of the expression of Cpf1 through the use of arabinose.
BBa_K5149102 PphlF Promoter (BBa_K1725000), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010) A genetic transcription unit that consists of promoter, Cas12a block, and terminator. The promoter pPhlF expresses Cas12a in the presence of a de-repressor DAPG.
BBa_K5149103 J23101 Promotor (BBa_J23101), GFP (BBa_K5149008), and rrnB T1 Terminator (BBa_B0010) A genetic transcription unit that consists of promoter, Green Florescent Protein (GFP), and a terminator. The J23101 is a strong promotor that will express many GFP units. We use this composite part as an insert into our organisms chromosomes so that our organism flouresces green.
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Software Contribution

The immune systems of L. fusiformis and other cyanobacteria are often poorly understood and vary by strain, making it difficult to predict how native systems will interfere with gene inserts. A common defense mechanism in L. fusiformis and UTEX 3154 is the Restriction Modification System (RMS), which cuts specific DNA sequences. This likely contributes to the degradation of genetic inserts, hindering transformation. Our PI, David Bernick, wrote the Stealth algorithm,[2] a program that identifyies underrepresented K-mer sequences in a given genome- we used the Stealth algorithm as a starting point to help solve the problem.

BLACKBIRD[1] uses the genomes of the host organism, the organism from where the gene insert is derived, and the target organism to optimize the gene insert. It calculates the codon usage table for the genomes. This is used to generate a ranking table between the host and target organism, so that when codon optimizing the sequence, codons are used in accordance with their relative abundance. This is done to preserve slow translating regions of proteins, shown to increase the folding efficiency of proteins as the time taken for the rarer tRNA to bind promotes proper protein folding [3][4]. The codon usage table is created through the use of a Open Reading Frame finder coded into the program, and generates the usage statistics based off of the frames found. When these are completed, the codon tables of both organisms are compared, matching codons to amino acids based on usage rankings between organisms, and the gene insert can be altered.


Learn more about our software, BLACKBIRD >>

Troubleshooting Contribution

Our DBTL (Design-Build-Test-Learn) cycles, as thoroughly documented on our wiki, serve as invaluable resources for teams looking to enhance their troubleshooting skills through experimentation. Each phase of the cycle—whether it’s the design, construction, testing, or reflection on outcomes—offers insights that teams can leverage to systematically address challenges and refine their processes. By making sure that our learning process and outcomes are easily accessible to others within the organization, we can actively contribute to bridging gaps in existing literature and collective knowledge. This becomes especially important when other teams are working with similar organisms, accelerator systems, or experimental setups as ours, as they can draw directly from our documented experiences to inform their own work. The transparency and availability of these shared learnings not only promote efficiency but also foster a collaborative environment where continuous improvement is encouraged across projects. Ultimately, this shared knowledge helps to reduce redundancies, accelerates innovation, and ensures that small, often overlooked issues are identified and resolved more quickly. Furthermore, as a part of our documentation, we also included troubleshooting tip on each protocol that was used in this project.


Learn more about the experiments we ran >>

References

[1] V. Nandakumar, A. Mahesh BLACKBIRD GitLab iGEM UCSC 2024.

[2] S. Hu, "Altering under-represented DNA sequences elevates bacterial transformation efficiency" mBio, Oct. 31, 2023. https://doi.org/10.1128/mbio.02105-23 (accessed Sep. 24, 2024).

[3] G. Zhang, "Transient ribosomal attenuation coordinates protein synthesis and co-translational folding" Nat Struct Mol Biol, Jul. 13, 2008 https://www.nature.com/articles/nsmb.1554 (accessed Sep. 26, 2024).

[4] G. L. Rosano, "Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain" Microb Cell Fact, Jul. 24, 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2723077/ (accessed Sep. 26, 2024).