Part:BBa_K1088018

LacI repressor from E. coli

Coding region for the LacI protein. LacI binds to the lac promoter BBa_R0010 and inhibits transcription. IPTG binds LacI and inhibits its function, and thus promotes transcription from the lac promoter.

FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088026) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088009) (lacI:LVA) were grown from OD₆₀₀ 0.005 to approximately 0.2. At this OD the one triplicate of each strain were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min. A) lacI(lva) and lacI(N) strains grew at same pace both with and without induction. B) Per cent of population above fluorescence threshold. Both lacI(lva) and lacI(N) represses the expression, when not induced with IPTG. lacI(lva) reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell per cent fluorescent compared to lacI(N). lacI(N) reaches a maximum just below 100 % at a time between 30 and 60 min. C) Mean GFP fluorescence of entire population. These results reflect what is seen in B, and clearly indicates that overexpression of natural LacI instead of LacI:LVA is the better, when a quick response and high level of expression is wanted.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Protein data table for BioBrick BioBrick-AutoAnnotatorNucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGAAACCA ... AGCGGGCAGTGA  ORF from nucleotide position 1 to 1080 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVE RSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAG WHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTS VDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence
Amino acid composition: Ala (A) 44 (12.2%) Arg (R) 19 (5.3%) Asn (N) 12 (3.3%) Asp (D) 17 (4.7%) Cys (C) 3 (0.8%) Gln (Q) 28 (7.8%) Glu (E) 15 (4.2%) Gly (G) 22 (6.1%) His (H) 7 (1.9%) Ile (I) 18 (5.0%) Leu (L) 41 (11.4%) Lys (K) 11 (3.1%) Met (M) 10 (2.8%) Phe (F) 4 (1.1%) Pro (P) 14 (3.9%) Ser (S) 32 (8.9%) Thr (T) 19 (5.3%) Trp (W) 2 (0.6%) Tyr (Y) 8 (2.2%) Val (V) 34 (9.4%)
Amino acid counting Total number: 360 Positively charged (Arg+Lys): 30 (8.3%) Negatively charged (Asp+Glu): 32 (8.9%) Aromatic (Phe+His+Try+Tyr): 21 (5.8%)	Biochemical parameters Atomic composition: C1684H2762N484O524S13 Molecular mass [Da]: 38590.2 Theoretical pI: 6.39 Extinction coefficient at 280 nm [M-1 cm-1]: 22920 / 23108 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.79) good (0.78) acceptable (0.55) good (0.60) good (0.78) good (0.66)
Alignments (obtained from PredictProtein.org) SwissProt: P03023 (100% identity on 360 AAs), Q9CN88 (32% identity on 325 AAs) TrEML: D8E1B5 (100% identity on 359 AAs), E3PES2 (100% identity on 360 AAs), C6EJI7 (100% identity on 359 AAs), Q3Z584 (100% identity on 360 AAs), C6UB29 (100% identity on 360 AAs), C6ZCU4 (100% identity on 360 AAs), C9QQT3 (100% identity on 359 AAs), G2F6I6 (100% identity on 360 AAs), G2CUK7 (100% identity on 360 AAs), G2CFY7 (100% identity on 360 AAs) PDB: 1lbg (99% identity on 357 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets) Archaea: cytosol (100%) Bacteria: cytosol (99%) Eukarya: mitochondria (61%)	Gene Ontology (reliability in brackets) Molecular Function Ontology: GO:0043565 (25%), GO:0003700 (29%) Biological Process Ontology: GO:0006355 (34%), GO:0045449 (18%)
Predicted features: Disulfid bridges:  - Transmembrane helices:  -
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