Chris M. Thomas

[17] Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2

Publisher Summary This chapter discusses the cloning vehicles related to plasmids ColE1, F, R6K, and RK2. Because these replicons have different properties, cloning vehicles derived from one of them may be more suitable for a particular application than vehicles derived from another. Also, because derivatives of a single plasmid do not stably coexist, it is often desirable to have available cloning vehicles from a variety of incompatibility groups to study the interactions among cloned fragments. The ColE1 vehicles pMK20, pMK16, and pMK2004 are very useful for general-purpose cloning because they are maintained in high copy number and provide good selective markers for transformation. Like the ColE1 vehicles, pRK353, a derivative of the antibiotic resistance plasmid R6K, is present in a large number of copies per cell and can be used as a high-copy-number cloning vehicle that can coexist with ColE1. In addition, the R6K replicon has been separated into two components that make up a functional replicon. Plasmid RK2 is unusual because it is maintained in a wide variety of gram-negative bacteria, and therefore RK2 derivatives may be of use as cloning vehicles in gram-negative bacteria other than Escherichia coli ( E. coli ).

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.

SummaryA DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.

Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2

SummaryReplication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small oriRK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus ‘incompatible” with RK2 replicons. However, the 393 bp oriRK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on oriRK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII oriRK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from oriRK2is required or may simply reflect the strong selective pressure for establishment of the incoming oriRK2 plasmid and poor ability of the HpaII oriRK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII oriRK2 may be designated inc1. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc2 region, to distinguish it from inc1, above, that overlaps but does not coincide with the 393 bp HpaII oriRK2. This inc2 region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete oriRK2. Also, only part of the inc2 region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for oriRK2.

Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

Abstract It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, oriRK2, located at 12 kb on the genome, is also required. A segment of DNA containing oriRK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from oriRK2. Deletion of the trfB region in trans to an oriRK2 plasmid does not correlate with alteration in copy number or stability of the oriRK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent oriRK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the oriRK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from oriRK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, oriRK2, trfA, and trfB.

Broadband Synthetic Transmission-Line N-Path Filter Design

A synthetic transmission-line N-path bandpass filter technique that lowers the in-band insertion loss and increases the out-of-band rejection of the traditional N-path filter is analyzed. The performance limitations of the traditional N-path filter in terms of in-band insertion loss and limited rejection are reviewed. Measurement results from 0.1 to 1.6 GHz of a single-ended, four-stage synthetic transmission-line N-path filter fabricated in a 65-nm CMOS technology that achieves less than 5-dB insertion loss across the tuning range, 30-50-dB out-of-band rejection, in-band third-order distortion input-referred intercept point (IIP3) of +29 dBm, and a +11-dBm input-referred 1-dB compression point (P1dBJam) out-of-band jammer tolerance are presented.

A 1.3 mW 48 MHz 4 Channel MIMO Baseband Receiver With 65 dB Harmonic Rejection and 48.5 dB Spatial Signal Separation

A four-channel multi-input multi-output (MIMO) complex baseband receiver for spectrum and space-aware cognitive radio applications is presented. The MIMO baseband receiver comprises a capacitive harmonic-rejection downconverting mixer (HRM) receiver and a signal-separation multi-input multi-output analog core (MAC) on a single integrated circuit. The HRM receiver performs frequency selection of the incoming RF signals by programmable spectral downconversion and filtering with minimal harmonic folding. The subsequent MAC separates the spectrally overlapping but spatially diverse signals by weighted complex matrix multiplication. The entire signal path is implemented using energy-efficient gm-C analog circuits with digitally controlled capacitive weighting for configurable baseband down-/upconversion ranging from -24 to +24 MHz in the HRM, and programmable spatial filtering with 4×4 complex (8×8 real) 14-bit coefficients in the MAC. Measurements demonstrate greater than 65 dB harmonic-folding rejection by the HRM, and greater than 48.5 dB spatial signal separation by the MAC. The 65 nm CMOS IC occupies 3.27 mm2 active area, and consumes 480 μW digital power at 45 MHz LO and 840 μW analog power at 3 MHz baseband from a 1.2 V supply.

A CMOS 4-channel MIMO baseband receiver with 65dB harmonic rejection over 48MHz and 50dB spatial signal separation over 3MHz at 1.3mW

A CMOS integrated 4-channel capacitive harmonic rejection baseband receiver and 4×4 MIMO analog core spatial filter demonstrate >65dB harmonic folding rejection over 48MHz, and >48.5dB signal separation across 3MHz baseband. The 65nm CMOS IC occupies 3.27mm2 active area and consumes 0.67mW-1.28mW.

A 65 nm CMOS Tunable 0.1-to-1.6 GHz Distributed Transmission Line N-Path Filter with +10 dBm Blocker Tolerance

A distributed transmission line N-path bandpass filter is presented from 0.1 to 1.6 GHz with 30 MHz 3dB pass-band bandwidth, LO leakage <; -60 dBm, 30 dB to 50 dB out-of-band rejection, in-band IIP3 of +23 dBm, and a +10.1 dBm out-of-band jammer tolerance in a 65 nm CMOS process.

High-Performance Silicon-Based RF Front-End Design Techniques for Adaptive and Cognitive Radios

Monolithic silicon-based RF front-end circuit approaches are presented as an alternative to traditional off-chip SAW and switch-based techniques, with the goal of implementing a fully integrated RF front-end. Tunable filters based on N- path techniques have demonstrated excellent noise and linearity properties, and are expected to further improve as CMOS technology continues to scale. Integrated duplexers for frequency division duplex wireless standards have also been developed recently, based on a broadband hybrid transformer approach.

A CMOS Broadband Distributed

A distributed CMOS N-path tunable band-pass filter (BPF) technique is introduced to lower the in-band insertion loss and increase the out-of-band rejection of the traditional N-path filter. Measurements from 0.3 to 1.6 GHz demonstrate a tunable BPF with 31 to 53 dB out-of-band rejection in a 65 nm process.