Improvement of co-electroporation and plasmid segregation efficiency through culture time decrease

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Different vectors for the Lambda-Red system

For Red recombineering strategy, in order to obtain the highest recombineering efficiency, an appropriate expressed level of the Red recombination proteins is required. Limited expression of Gam protein couldn’t inhibit the RecBCD nuclease activities completely (Sergueev, Yu et al. 2001), a high expression of Gam could also cause plasmid instability (Silberstein Z 1990; Murphy 1991) and even cell death (Kirill Sergueev 2001). The Red enzymes could be expressed under the control of their own promoter or under heterologous regulated promoters. While endogenous phage promoter leads to tight regulation and coordinates expression thus leading to a higher recombination frequency (Court DL 2002). Court et al (Yu, Ellis et al. 2000; Ellis, Yu et al. 2001; Court DL 2002) have developed and utilized a prophage Red system on the E.coli chromosome for recombineering application. In this system, they do some genetic modification of prophage including the deletion of lysis, replication and structural genes thus inactivate the prophage infectious cycle. However, the critical functions such as transcriptional control of the Red are retained (Fig.28).
Fig.28: Schematic representation of λ phage constructs into E.coli chromosome used for recombineering (Sharan, Thomason et al. 2009) Bacterial DNA is shown in blue bar, phage DNA in red bar and the region containing a deletion and/or a substitution is a white bar. (a) Genetic structure of λ prophage which is integrated into the bacterial chromosome: the complete λ prophage is flanked by its attachments site att, where integration with the bacterial chromosome occurred and is adjacent to the bacterial biotin genes, bioAB. The int and xis genes located in the pL operon and encode function products which can help phage DNA to integrate into or excise phage DNA out of the bacterial chromosome. There are two operons as shown with arrows and their promoters pL and pR in the prophage. The exo, bet, and gam genes are under the control of the temperature-sensitive repressor CI857. At temperatures less than 32°C (Yu, Ellis et al. 2000), CI repressor binds to t he operators and prevents transcription of the pL operon and thereby the expression of Red. The expression of the Red system can be turned on easily at 42°C inducing the repressor CI857 denaturation and as a consequence, al lows the Red genes transcription. When shifting back to low temperature, CI857 re-natures and recovers its initial functions thus completely blocks the transcription of pL. When λ Red functions are turned on for a short time period, like 5 min, cells become more recombinogenic after linear DNA uptake (Yu, Ellis et al. 2000). The N gene encodes an anti-transcription terminator and prevents RNA polymerase termination of pL transcripts at terminators tL1, tL2, and tL3. The kil gene is adjacent to gam and when expressed for over 1 hour kills the host bacterial cell (Sergueev, Yu et al. 2001). The replication genes O and P of the λ prophage are in the pR operon. Cro functions as a partial repressor of the pL and pR operons when CI is inactive at 42°C. The lysis and structural genes, SRA-J, are shown located beyond their regulator Q (Sharan, Thomason et al. 2009).
(b)The defective prophages used for recombineering in DY380, SW102, and the HME strains (Lee, Yu et al. 2001; Warming S 2005): cro through the bioA prophage genes are deleted. The right att site is also deleted preventing any excision of the prophage. In DY380 and SW102 strains, those deleted genes are replaced by the tetracycline resistance cassette, tetRA.
(c) The mini-λ phage DNA (Court, Swaminathan et al. 2003) is shown with the lytic genes cro through J deletion. In different constructs of the mini λ, the cro-J region is replaced with various drug resistant cassettes.
As we mentioned before in the genetic structure of λ prophage, mini- λ has kept att sites plus int and xis, allowing its integration and excision.
Recently, for the convenient use of Red systems, its critical elements were integrated into a set of different plasmids. For instance, Murphy et al (Murphy 1998; Murphy, Campellone et al. 2000) designed a plasmid possessing phage recombination functions under the control of an IPTG-inducible lac promoter. Later on, Datsenko (Datsenko and Wanner 2000) and Wang (Wang, Sarov et al. 2006) developed recombineering plasmids under the arabinose inducible pBAD promoter (Chao, Chiang et al. 2002; Terpe 2006). In this construction, the AraC protein is both a positive and a negative regulator (Yajima, Muto et al. 1993; Cobb and Zhao 2012). In the presence of arabinose, transcription from the pBAD promoter is turned on; in its absence, transcription occurs at very low levels (Lee, Francklyn et al. 1987). Other studies (Dodd, Shearwin et al. 2005; Donald L. Court 2006) show that the repression system of the Red machinery is strong exclusively under the controls of pL and pR promoters. So the ideal conditions is to ensure the presence of pL and pR promoters on all of the prophage constructs, whether carried on the bacterial chromosome, on low copy plasmids or under the control of a temperature sensitive gene such as cI857.
The pSIM vectors (Datta, Costantino et al. 2006) consist of the basic elements for its replication and other necessary elements for prophage induced homologous recombination. Fig.28 (d) shows the segment of the pSIM plasmids. The genes from cro to att and genes beyond tL3 including int and xis were removed. The red genes were directly connected to pL by a deletion that removes kil through N genes. The drug resistance marker specific of each pSIM plasmid replaces the rex gene (inhibit cell function without phage superinfection (L snyder 1989) ) adjacent to cI857. The basic features are conserved in all of these Red expression pSIM plasmids.
This Red system was left under the control of native phage elements for optimal expression and
regulation. The pL promoter drives gene expression and is controlled by the temperature sensitive but reversible CI857 repressor. In all, the cro gene is inactive to maximize pL expression, and the replication genes are absent or inactive to prevent lethal effects on the cell.

Substrates for the Lambda-Red system

Based on the strategy of Red system, different DNA substrates could be used. Exogenous double or single-strand linear DNA both need to contain two regions homologous to their target sequences in any case. The inserted region between two highly conserved sequences could either be a selective marker or a non-selective DNA (Roberts 2005). Yu et al. (Yu, Ellis et al. 2000) demonstrated that 30- to 50-bp homologies are able to result in recombination in vitro. For those exogenous DNA substrates constructions, we only need to add homology sequences (30- to 50-bp) at each 5’ end of PCR primers. These primers can amplify the exogenous DNA with highly homology conserved sequences easily at each end of PCR products and this PCR dependant DNA substrates preparation could substantially facilitates the Red system application because we can modify the homology sequences as we want.

Double-strand DNA recombination

ds DNA substrate can be generated by polymerase chain reaction (PCR) with a pair of primers containing homologous sequences to the target site (Arber and Linn 1969). Each primer, bout 70 bases in length, contains 50 nucleotides at the 5’ end corresponding to the homologous targeted region and around β0 nucleotides at its γ’ end to amplify the exchanged sequences (Fig.29).

Table of contents :

Part I: quinoline biodegradation in soil microcosm
1. Bibliographical review of quinoline and its biodegradation
1.1 Quinoline biodegradation
1.1.1 Pseudomonas sp. for quinoline biodegradation
1.1.2 Rhodococcus sp. for quinoline biodegradation
1.1.3 Comamonas sp. for quinoline biodegradation
1.1.4 Thauera.sp for quinoline biodegradation
1.2 Metabolic quinoline pathway
1.2.1 Metabolic quinoline pathway by Pseudomonas.sp
1.2.2 Metabolic quinoline pathway by Rhodococcus.sp
1.2.3 Metabolic quinoline pathway under anaerobic/anoxic condition
1.3 Genes involved in the quinoline biodegradation
2. Materials and methods
2. 1 Quinoline microcosm
2.2 RISA (rRNA intergenic spacer analysis)
2.3 GC/MS analysis for quinoline biodegradation
3. Results
3.1 Nucleotide BLAST of bcr operon in the Rothamsted metagenome
3.2 RISA results
3.3 Results of GC/MS analysis
4. Discussion and conclusion
PART II: in vitro development and use of Genefish to capture targeted DNA fragments
1. Background
2. Bibliographical review of Lambda-Red homologous recombination and Genefish approach
2.1 Lambda-Red homologous recombination
2. 1.1 Introduction
2.1.2. Lambda-Red recombination system
2.1.2.1 Overview of Lambda-Red recombination system
2.1.2.2 Different vectors for the Lambda-Red system
2.1.2.3 Substrates for the Lambda-Red system
2.1.2.3.1 Double-strand DNA recombination
2.1.2.3.2 Single-strand DNA recombination
β.β. The “Genefish” tool
β.β.1 General presentation of “Genefish”
2.2.2 The suicide cassette
2.2.3 Homologous recombination with Lambda-Red system
2.2.4 bcr operon
3. Genefish application using bcr operon
3.1. Capture plasmid and host strain construction
3.1.1 Materials:
3.1.2 Methods:
3.1.2.1 Highly conserved bcr fragment sequences determination
3.1.2.2 Capture plasmid construction
3.1.2.3 Host strain construction
3.1.2.4 Escape rate test
3.1.3 Results
3.1.3.1 Highly conserved bcr fragment selected for capture plasmid construction
3.1.3.2 Capture plasmid construction
3.1.3.3 Host strain construction and escape rate test
3.2. Genefish tool application
3.2.1 Materials
3.2.2 Methods:
3.2.2.1 Co-electroporation
3.2.2.2 Plasmid segregation
3.2.3 Results.
3.2.3.1 Co-electroporation by using bcr c-d fragment
3.2.3.2 Plasmid segregation by using pBAD35K7toxN-bcrc-d
3.2.3.3 Plasmid segregation by using pBAD35K7toxN-bcrc-a
3.3. Genefish improvements
3.3.1. Materials
3.3.2 Methods:
3.3.2.1 Improvement of co-electroporation and plasmid segregation efficiency through culture time decrease
3.3.2.2 Single-copy plasmid construction
3.3.2.2.1 Classical digestion and ligation
3.3.2.2.2 In-Fusion HD Cloning Kit method
3.3.3 Results:
3.3.3.1 Improved co-electroporation
3.3.3.2 Improved plasmid segregation
3.3.3.3 single-copy capture plasmid construction
4. Conclusions and Discussion
5. Perspectives

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