Development of a New Integration Site within the Bacillus subtilis
Chromosome and Construction of Compatible Expression Cassettes
B. Härtl et al. (2001)
J. Bacteriol. 183: 2696-2699
Outline
1. Introduction
2. Objectives of the Experiments
3. Experiments
3.1 Construction of the lacA delivery expression vector pAX01
3.2 Construction of a lacA::spec chromosomal insertion mutation
3.3 Construction of the IPTG-inducible expression vector pA-spac
Outline
3.4 Regulation of the Expression of β-Gal from the lacA locus by the integrative expression vectors pAX01 and pA-spac
3.5 Integration of hrcA gene at the lacA locus and Regulation of the Expression of an inserted gene at the amyE locus
4. Conclusions
5. Take – Home – Lesson
6. Discussion
Introduction
• several different plasmids as cloning vectors in B. subtilis
→ disadvantage: replication via single-stranded DNA intermediate
→ instability of these plasmids
• alternative method: usage of integrative plasmids
→ widely used system: nonreplicating delivery vectors
Introduction
• ectopic integration: insertion of a foreign gene at a non-homologous site
• simplest form of the system:
→ antibiotic resistance marker gene + MCS enclosed by two halves of the amyE gene (amyE-front & amyE-back)
→ tranformation into B. subtilis cells
→ recombination at homologous sites via double- crossover
Objectives
• Generation of an additional integration site at the lacA locus in Bacillus subtilis
• Development of the two different delivery expression vectors pAX01 and pA-spac
• Creation of the two recipient strains IHA01 and IHA02
• Verification which of the both vectors works superior in comparison to each other
• Demonstration of the communication between the two loci lacA and amyE
Construction of the lacA delivery expression vector pAX01
Three different fragments were produced: 1. lacA-front and lacA-back (generated seperately)
→ template: chromosomal DNA of B.subtilis 2. xylose expression cassette fused to the transcription
terminator → templates: pX and phage DNA 3. Erythromycin resistance gene (erm) → template: pMUTIN4 Assembling of the fragments by ligation with the usage of
restriction enzymes → pAX01
Construction of the lacA delivery expression vector pAX01
• modular structure of the delivery expression vector
→ greatly improved versatility
→ ability to adjust it to all sorts of applications
Construction of a lacA::spec chromosomal insertion mutation
• Integration of pAX01 can occur via single- or double-crossover event
→ single-crossover: integration of the whole vector at the lacA locus
→ double-crossover: only integration of the desired gene
→ Ermr - transformants
Construction of a lacA::spec chromosomal insertion mutation
• Discrimination of these transformants:
→ creation of a recipient strain that contains a Specr marker in the chromosomal DNA at the lacA locus
IHA01 (lacA::spec)
• previously described recipient strain for the amyE locus: AM01 (amyE::cat)
Construction of a lacA::spec chromosomal insertion mutation
• Combination of lacA::spec and amyE::cat in a single strain:
→ preparation of chromosomal DNA from IHA01 and transformation into AM01
→ IHA02
→ allows insertion of two different DNA sequences at
both loci within the same strain
Construction of the IPTG-inducible expression vector pA-spac
• E. coli lac promoter system as the most widely used expression system in B. subtilis
→ negative control by lac-repressor
→ IPTG as an inducer (inactivates repressor)
• Construction of the pA-spac vector:
→ creation of a lac expression cassette (includes lacI gene and lac promoter-operator region)
→ insertion into pAX01 → pA-spac
Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other
• There are two expression cassettes that would be compatible for a new integration site within the B. subtilis chromosome
→ pAX01 and pA-spac
Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other
• Question: Which of both vectors works superior?
1. insertion of a reporter gene (bgaB; encodes for heatstable β-Gal) into both vectors
→ pAX01-bgaB and pA-spac-bgaB
2. transformation into IHA01
→ IHA01-Xyl-bgaB and IHA01-Spac-bgaB
3. measurement of the activity of the enzyme in absence and in presence of an inducer
Regulation of the Expression of β-Gal using the vector pA-spac
• presence of IPTG:
→ no influence on the growth if the strain
→ steady increase of the activity of β-Gal over time
→ 0,1 mM IPTG was sufficient to reach full induction rate
• absence of IPTG:
→ slow increase of the activity
→ there is some leakiness in the system
Regulation of the Expression of β-Gal using the vector pAX01
• presence of Xylose:
→ no influence on the growth if the strain
→ high increase of the activity of β-Gal
→ decline after 200 min (consumption of the sugar)
→ 0,5 % Xylose was sufficient to reach full induction rate
• absence of Xylose:
→ no activity
Verification which of the both vectors (pAX01 and pA-spac) works superior in comparison to each other
The Xylose – inducible system is superior to the
IPTG – inducible system because of two reasons:
1. There is no expression of the bgaB gene in the absence of the inducer xylose
2. It results in a higher inducction rate
(40 – folds versus 15 – folds)
Communication between the two loci lacA and amyE
• amyE locus existed already as an integration site
• lacA locus was developed as a new integration site
• Question:
Is this new integration site useful in obtaining regulated expression of a gene that was integrated at the amyE locus in the same strain?
Communication between the two loci lacA and amyE
• creation of a strain that contains at the lacA locus a gene for a repressor affecting the operator of the gene that was integrated at the amyE locus
→ generation of the hrcA repressor gene and ligation into pAX01 → pAX01-hrcA
→ transformation into AM01 and homologous recombination (AM01: no own hrcA gene + reporter gene bgaB at the amyE locus ) → IHA13
Communication between the two loci lacA and amyE
• Measurement of the β-Gal activity
• Absence of Xylose:
→ no expression of the hrcA repressor gene
→ high expression of the bgaB gene
→ increase of the β-Gal activity
• Presence of 0,5 % Xylose:
→ decay of the activity to about 10 U
→ about 3 h after the addition of the inducer the β- Gal activity began to rise
Communication between the two loci lacA and amyE
Result:
Regulated expression from one chromosomal locus can influence the transcription of a distant one.
Conclusions
• lacA locus as a new integration site for ectopic integration
→ codes for very weakly expressed β-Gal • lacA::spec as a chromosomal insertion mutation in the
IHA01 strain (for easy screening) • creation of two delivery expression vectors:
pAX01 & pA-spac • measuring the activity of the β-Gal results in the fact
that pAX01 is the better vector for regulative expression
• the expression of the hrcA gene at the lacA locus was able to affect the expression of the bgaB reporter gene at the amyE locus
Take – Home - Message
The lacA locus was developed as a new site fo ectopic integration within the B. subtilis chromosome and the integration of a regulatory gene at this site is able to
control the expression of a transcriptional fusion at the amyE locus.