Left end of a linear transpososome sequence containing the 19 bp mosaic end in the forward orientation. The sequence also contains a PvuII cut site which releases the transpososome when cut with PvuII. Constructed compound parts intended for transposition must not contain other PvuII end, or be PCR amplified with blunt end producing enzymes (Pfu, Phusion) at the PvuII cut site between CAG and CTG. This part, and the companion part M1001 provide Biobrick tools to construct Tn5 transpososomes: linear DNA sequences which can be cut in vitro from a plasmid backbone, or created by PCR, combined in vitro with purified Tn5 trasposase protein, electroportated into cells, and finally randomly inserting into the bacterial chromosome. The parts assembled between the two mosaic ends can be delivered, including antibiotic resistance markers or other functional chromosomal insertions.
;biobrick left end, TA cloning overhang GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ;biobrick right end, TA cloning overhang GTT TCT TCC TGC AGC GGC CGC TAC TAG TA ;mosaic end with cag upstream to insert PvuII cut site cagCTGTCTCTTATACACATCT ;mosaic end reverse complement with ctg upstream to insert PvuII cut site AGATGTGTATAAGAGACAGctg ;mosaic left end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG cagCTGTCTCTTATACACATCT, MEL-F ;mosaic left end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA AGATGTGTATAAGAGACAGctg, MEL-R ;mosaic right end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG AGATGTGTATAAGAGACAGctg, MER-F ;mosaic right end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA cagCTGTCTCTTATACACATCT, MER-R
Upstream genomic DNA fragment of Mesoplasma florum for recombination experiment
Approximately 450 bp upstream DNA fragment of M. florum genome from sites 352951-353409 intended as a recombination site for homologous recombination, removing the restriction enzyme and methylase from M. florum.
Avoidance of biobrick restriction sites, possible primer binding sites.
PCR from the M. florum genome. Primers: * ttccgaattcgcggccgcttctagag tccaaatagtgtggatac,Mfl2I-up-F * ttccactagtaaagacatctatgtttttttac,Mfl2I-up-R
Plasmid origin from pWV01 + Tet resistance gene for plasmid construction
Plasmid origin from pWV01 + Tet resistance gene for plasmid construction. Intended to be cut with XS and inserted into the NheI sites of vector construction part I15020.
Avoid assembly into tet resistant plasmids since it already has tet resistance.
Downstream genomic DNA fragment of Mesoplasma florum for recombination experiment
Recombination site downstream of the restriction enzyme and methylase of M florum. 700 bp fragment between 356063-356763.
no biobrick restriction enzyme sitss, primer binding
PCR from the M. florum genome. Primers: * ttccgaattcgcggccgcttctagag gagattattatggcaaaag,Mfl2I-dn-F * ttccactagtagtaatatgagtaaattcac,Mfl2I-dn-R
Novel gentamicin resistance gene. Derived from Genbank DQ208936, pTEX5500ts, kind gift of Barbara E. Murray. This, in turn was derived from Genbank AF016483. See pmid=16391062, pmid=9593155
XbaI site at 660 removed by mutating c->t which also improves codon usage. gatgatttttacagggatattttagagaacgaaatctac,P1014-XF gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR
recT recombination gene from Spiroplasma citri
recET recombination gene from Spiroplasma citri, probably catalyzing the homologous recombination of DNA fragments similar to the Lambda Bet gene (Lambda RED recombination). This gene is from S. citri, a close relative of Me. florum, and has very similar codon usage.
Eliminate GATC site by silent mutation to GACC (asp -> asp). Note very low GC content at the 5' end. The primers are: * gtttcttcgaattcgcggccgcttctagatgaataataaaattgaattaatattaaataattc,recT-F * gtttcttctactagtagcggccgctgcagttattaatcagaaaacatttcattaacagc,recT-R * gaaatgcaagaagcctatcaacatgaccaagcaataattattaataacagc,recT-mut-F * gctgttattaataattattgcttggtcatgttgataggcttcttgcatttc,recT-mut-R
Genbank entry CAK99381 protein, nucleotide coding region. Mutations of the single GATC site to avoid restriction by the Sau3AI restriction system in Me. florum. PCR from S. citri DNA, kind gift of Prof. J. Renaudin.
Assembly of tetM cassette and pSCI2 plasmid origin into E. coli plasmid pSB1K3.
Assembled into Kan resistant part from cm resistant parts. Avoid tet resistant backbone plasmids, since the insert is tet resistant.
Plasmid pBG7AU (J70310) for Mesoplasma florum combined with Tet resistance cassette (J70028).
Right end of a linear transpososome sequence containing the 19 bp mosaic end in the reverse orientation. The sequence also contains a PvuII cut site which releases the transpososome when cut with PvuII. Constructed compound parts intended for transposition must not contain other PvuII end, or be PCR amplified with blunt end producing enzymes (Pfu, Phusion) at the PvuII cut site between CAG and CTG. This part, and the companion part M1000 provide Biobrick tools to construct Tn5 transpososomes: linear DNA sequences which can be cut in vitro from a plasmid backbone, or created by PCR, combined in vitro with purified Tn5 trasposase protein, electroportated into cells, and finally randomly inserting into the bacterial chromosome. The parts assembled between the two mosaic ends can be delivered, including antibiotic resistance markers or other functional chromosomal insertions.
Synthesis from overlapped oligos with PCR.
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F2 to OR-R4 GTTTCTTC T TCTAGA G ttttgatggttgtcaagttc,OF-F2 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A cctgtaatgctgggttaac,OR-R4
Needs mutation of EcoRI sites and GATC sites.
constitutive cat cassette for Mesoplasma florum
This part is a version of J70026, the cat cassette from pC194. The original cassette is inducible, with a 5' UTR containing a hairpin structure which sequesters the Shine-Dalgarno sequence. In this mutation, we eliminate the hairpin structure from the 5' UTR to attempt a constitutive construct.
Test for KpnI sites in the source plasmid.
PCR from pSB1T3-J70026 with primers GTTTCTTCggtacctttcttcattagaatcaatcc,cat-mut-R GTTTCTTCggtacctttaggaggcatatcaaatg,cat-mut-F These primers bind to the source plasmid and remove the left side of the hairpin and add a pair of KpnI sites, which will be cut and religated. I hope to select on Cm plates, since the construct should be Cm resistant.
MF optimized RFP reporter, MF heat induced promoter
RFP optimized for expression in E. coli and M. florum
Codon optimized version of red fluorescent protein, optimized for dual expression in E. coli and M. florum. Includes an RBS and CDS. Designed to express well in either species. Designed with Gene Designer and made by DNA 2.0, provided in their pJ201 plasmid with Kan resistance.
Restriction site elimination, coding bias, elimination of CGG codon.
Tet resistance + Mesoplasma origin fragment
Promoter from the highly expressed pdh e1 gene (mfl039) of Me. florum. Contains three overlapping promoters with near consensus sequence and an UP sequence.
53556 53678 approximately from NC_006055
TetM gene, recoded compromise E. coli & Mesoplasma florum
The tetM antibiotic resistance gene synthesized by Geneart from a protein sequence derived from the Ureaplasma urealyticum tetM gene. Synthesized with a compromise codon usage table for expression in both E. coli and Mesoplasma florum. Highly active in both E. coli and Mesoplasma florum.
Elimination of restriction sites, CGG codons, UGA codons, rare codons in both species.
Synthesized from the Ureaplasma urealyticum tetM gene sequence, with recoding.
Promoter 43 and RBS spoVG for B. subtilis
Constitutive promoter 43(<bbpart>BBa_K143013</bbpart>) coupled to the strong Ribosome Binding Site spoVG(<bbpart>BBa_K143021</bbpart>) from ''B. subtilis''. P43-spoVG can be used in the context of a Polymerase per second (PoPS) output generator. '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of P43 and RBS-spoVG were obtained from papers1 2 and the sequence synthesised by GeneArt.
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F1 to OR-R4 GTTTCTTC T TCTAGA G aagtgctggaaaaagtatag,OR-F1 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A cctgtaatgctgggttaac,OR-R4
Heat shock inducible promoter for Me. florum
Promoter inhibited by the gram + hrcA gene product in normal temperature conditions. Promoter from the lon protease,
PCR from lon protease promoter of NC_006055.
pWV01 broad host range plasmid origin and rep protein
pWV01 broad host range plasmid origin and rep protein. pWV01 replicates by rolling circle replication in a very broad range of bactera, including both gram positive and gram negative species. This part is the a stripped down plasmid origin for constructing a Biobrick plasmid.
Avoided a single Sau3AI site near the claI site, to eliminate Sau3AI sites in the final plasmid assembly.
PCR of the plasmid pMG, kind gift from Prof. Jan Kok. This plasmid contains the ClaI fragment of the pWV01 plasmid.
gentamycin/kanamycin resistance cassette
The aacA-aphD gene from the transposon Tn4001 conveys resistance to both Gentamycin and Kanamycin with a single two-domain resistance protein.
;primers to make Biobrick version of GmR gene cassette (P1012) gtttcttcgaattcgcggccgcttctagagTGGACTTGACTGAGTTTATGGAA, P3aph-F gtttcttcctgcagcggccgctactagtaTCTGGACTTGACTCACTTCCTTT, P3aph-R ;P3aph-R (3' - 5') ;aaaggaagtgagtcaagtccagatactagtagcggccgctgcaggaagaaac
Originally from Tn4001 transposon; sequence M18086. Created from plasmid pISM2062 from Chris Minion, Iowa State University.
Click beetle luciferase derived from Promega's red/green system, modified using the E. coli/Mesoplasma florum compromise codon table on open wet ware. Removal of all restriction sites necessary to make fully Biobrick compliant.
Derived from Promega's click beetle luciferase red/green system, originally designed to function in mammalian cells. From the open reading frame of the pCBG99 vector.
TetM recoded for dual E. coli/Mesoplasma expression
This part is a cassette containing the PE gene from the pSci2 plasmid of Spiroplasma citri. It is reported to convey replication ability for the group 1 spiroplasmas. The part contains the replication origin, the promoter, and gene for the PE protein.
No native restriction sites. Added biobrick ends. The gene for PE contains no CGG codons. GTTTCTTCGAATTCGCGGCCGCTTCTAGAGtaattataaaaacaccttattttac,J00023-F2 GTTTCTTCCTGCAGCGGCCGCTACTAGTAttgggagtgcggggtttggggtgc,J00023-R The start of J70027 matches the origin start in the pSci1 plasmid.
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F2 to OR-R3 GTTTCTTC T TCTAGA G ttttgatggttgtcaagttc,OF-F2 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTATTAATTTTTCCTTCTTTAAC,OR-R2
J70025 (pBOT1 tetM promoter upstream region) + J70024 (recoded tetM gene from Geneart plasmid construction)
Test Cm resistance plasmid for Mesoplasma florum
Assembly of pSCI2 plasmid origin and cat cassette into E. coli plasmid pSB1T3.
Avoid assembly into a Cm resistant plasmid, as the construct is already Cm resistant.
Promoter for tetM gene, from pBOT1 plasmid, pAMbeta1
Upstream promoter for tetM gene; contains a short peptide leader, regulatory sequences which are poorly understood. Use in combination with J70024.
Alignment of the promoter with the coding region, preservation of the RBS sites.
pBOT1 plasmid PCR. pBOT1 from J. Renaudin. Original sequence is part of pAMbeta1 tetracycline resistance plasmid.
gentamycin resistance cassette (aacC1 gene)
The aacC1 gene conveys resistance to gentamycin only. It is derived from a Tn21 series transposon (Wohlleben89) found on the R1033 plasmid (Pseudomonas aeruginosa). This part is created by PCR from the pJAT8 plasmid (Keastling lab) which in turn is derived from pJN105 (Newman and Fuqua).
pJAT8 plasmid edited with primers: GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ccttgtcgccttgcgtataa,aacC1-F GTT TCT TCC TGC AGC GGC CGC TAC TAG TA atctcggcttgaacgaattg,aacC1-R
No native restriction sites. Added biobrick ends.
pheS gene from the phenylalanine tRNA charging complex in M. florum, preparing for mutation for use with p-chlorophenylalanine counter selection (Kast, pKSS).
PCR from M. florum genomic DNA. Primers: * ttccgaattcgcggccgcttctagag ctctgttattggtatagtta,pheS-mf-F2 * ttccactagtatgttctacctactctcc,pheS-mf-R
cat cassette (chloramphenicol resistance)
cat gene and promoter from pC194 plasmid, Genbank V01277.
Mutation in sequence at base 99, ignored. GATC site eliminated at the end by truncation.
Genbank V01277, by way of pKV104 from Duncan Krause. Bases 978-2006; missing T in the Krause sequence at base 99 of this sequence.
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F1 to OR-R3 GTTTCTTC T TCTAGA G aagtgctggaaaaagtatag,OR-F1 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTTAATCTCATATAACACCTC,OR-R3
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F3 to OR-R4 GTTTCTTC T TCTAGA G gaatttaaaaaaatagttgagtc,OR-F3 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A cctgtaatgctgggttaac,OR-R4
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F1 to OR-R2 GTTTCTTC T TCTAGA G aagtgctggaaaaagtatag,OR-F1 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTATTAATTTTTCCTTCTTTAAC,OR-R2
Mesoplasma florum origin of replication Section of Mesoplasma florum origin of replication from OR-F3 to OR-R3 GTTTCTTC T TCTAGA G gaatttaaaaaaatagttgagtc,OR-F3 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTTAATCTCATATAACACCTC,OR-R3
None. Modified base in the 5' end of the binding region of the F3 primer eliminates an EcoRI site.
RBS + mCherry gene, optimized for expression in Me. florum and E. coli
RBS + mCherry gene, optimized for expression in Me. florum and E. coli.
Initial tag of mCherry replaced with original AA sequence to eliminate the cryptic RBS + start internal to the coding sequence. Removed PvuII and Sau3AI sequences. Use of the compromise codon table guarantees no CGG codons and the use of TGG for tryptophan.
Synthesized by DNA 2.0, designed with Gene Designer. Initial tag of mCherry replaced with original AA sequence to eliminate the cryptic RBS + start internal to the coding sequence. Removed PvuII and Sau3AI sequences. Use of the compromise codon table guarantees no CGG codons and the use of TGG for tryptophan.
Section of Mesoplasma florum origin of replication from OR-F2 to OR-R3 GTTTCTTC T TCTAGA G ttttgatggttgtcaagttc,OF-F2 GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTTAATCTCATATAACACCTC,OR-R3
Needs mutation of EcoRI site and GATC sites.
Derrived from Promega's click beetle luciferase red/green system, originally designed to function in mammalian cells. From the open reading frame of the pCBR vector.
This is a biobricked version of the circular plasmid pBG7AU, Genbank NC_002569. The part is cut at the end of the Orf1 coding region, following the transcriptional terminator. This is similar to the location of the cut site used in the pKMK1 construction of Dybvig et al (King94) which formed their plasmid p2D4.
Unclear location for the cut site of the circular plasmid.
PCR from recircularized pBG7AU cut at the ClaI site. Original plasmid from Djordjevic01. Primers: GTTTCTTC GAATTC GCGGCCGC T TCTAGA Ggagtaaattctcctttttttgg,BG7AU-F GTTTCTTC CTGCAG CGGCCGC T ACTAGT Acaaaaaaaggagaatttactcc,BG7AU-R