CLONING IN GRAM POSITIVE BACTERIA E.g.: Bacillus Subtilis
Cloning in Gram-positive bacteria1.Characterization of the base composition of Gram-positive bacteria: <30% GC~>70%2. Preferred codons: Differ greatly from one organism to another.
Vectors for cloning in Bacillus subtilis and other low-GC organisms1. Characters of vectors: plasmids from S. aureus can be transformed into B. subtilis, but segregative stability are greatly reduced following insertion of exogenous DNA.2. Construction of vectors: Shuttle vectors for E.coli and another host cell
Many of the cloning vectors used with Bacillus subtilis and other low-GC bacteria are derived from plasmids found in Staphylococcus aureus• Development of B.subtilis with the observation (Ehrlich 1977) that plasmids from S.aureus can be transformed into B.subtilis, where they replicate and express antibiotic resistance normally.
Properties of some S.aureus plasmids used vectors in B.subtilis PLASMID Phenotype Size Copy no. Comments conferred on host cell pC194 Chloramphe 2906 bp 15 High mol wt nicol res. DNA pE194 Erthromycin 3728 bp 10 Temp res. sensitive for replication pUB110 Kanamycin 4548bp 50 Conjugal resistance transfer
• Because of the difficulties in direct cloning in B.subtilis hybrid plasmids were constructed which can replicate in both E.coli and B.subtilis• Most of the plasmid were constructed between Pbr322 and pC194,with such plasmids E.coli can be used as an efficient intermediate host for cloning.• Plasmid preparations extracted from E.coli clones are used to transform B.subtilis cells. Such preparations contain MCS.
1. Structural instability of recombinant DNA in B.subtilis: Longer DNA fragments often undergo rearrangements.2. Major reasons for structural instability : Mode of replication DNA fragments in plasmids replicating by a rolling-circle mechanism are likely to be unstable. While DNA fragments in plasmids replicating by theta mechanism tend to be stable
Transcription and translation1. The composition of the core RNA polymerase in B.subtilis and other low-GC hosts resemble that of E.coli and main sigma factor is sigma70, but the number is different.2. Many promoters contain TGTG motif at –16 region.
3. The ribosome of B.subtilis differ from that of E.coli in that B.subtilis ribosomes lack a counterpart of the largest E.coli ribosomes protein, S1.4. E.coli ribosomes can support the mRNA from other organisms, but B.subtilis ribosomes only support the homologous mRNA.
Controlled expression in B. subtilis and other low-GC hosts1. Controlled expression in B. subtilis : Plasmids contain T7 promoter and Lac operator as well as LacI, T7 RNA polymerase gene is inserted into B. subtilis chromosome DNA under the control of xylose-inducible promoter
2. Controlled expression in L.lactis : Low-copy plasmids contain a phage middle promoter and phage ori, the gene of interest is inserted into the downstream of the promoter. Following infection of φ31 phage, the plasmid number rapidly increases and the gene can be expressed in this way
Secretion vectors for low-GC bacteria1. The difference of signals between the low-GC bacteria and other organisms 1) The N-termini are more positively charged. 2) There are also larger and the extra length distributed among the three regions of the signal peptide.
Vectors for systematic gene inactivation1.Vector used for insertional mutagenesis in B.subtilis: pMUTIN2. Properties of pMUTIN 1) an ability to replicate in B.subtilis 2) a report gene to facilitate the measurement of the target gene 3) the inducible Pspac promoter
Properties of pMUTIN• An inability to replicate in B.subtilis, which allows insertional mutagenesis• A reporter lacZ gene to facilitate the measurement of expression of the target gene• The inducible Pspac promoter to allow controlled expression of genes downstream of and found in the same operon as the target gene