Team:UIUC-Illinois/Project

Track Selection: New Application

Our project, E. chiver, drew inspiration from the commonly used CRIM system, a series of plasmids that allows the user to integrate constructs into lambdoid phage sites common to many bacterial chromosomes. Our E. chiver system adds several elements yielding new applications. Our team designed two E. chiver constructs utilizing Lambda and P21 machinery. Each can in theory be used to shuttle a plasmid construct between two forms: a single chromosomal insert and a high copy number plasmid. In their current designs the systems must function separately, but possible routes have been identified by our team to make the co-functioning of these systems possible. We can see elements of our project being used in drug delivery systems as a method to keep a gene of interest dormant unless in the correct condition/location, and with further exploration into the co-functioning routes it may be used to create a ‘bacterial filing cabinet’.

To create a system that allows a construct to be expressed at a high copy number when induced, but then file back into the chromosome as a stable integrant for energetically favorable storage. The ultimate end goal is to create a bacterial filing system, E. chiver, which allows the coexistence of multiple constructs with their copy number and integration tied to a unique inducer.

A simple analogy for our E. chiver project is a filing cabinet. The following are the basic components of the system and their workplace analogues.

Construct = Shuttle Plasmid = File

Gene of interest = File Contents

Chromosome = Filing Cabinet

Luckily for us, the basic machinery we need for our E. chiver design already exists. The machinery is that of the Lambdiod phage family (i.e. λ, P21, P22, HK022, φ80), as well as the conditional R6K origin of replication and its trans-acting factor pir. The following are basic descriptions of how these parts function.

Lambdoid phage machinery: Our main interest is the phage proteins excisionase (Xis) and integrase (Int), as well as the phage genetic element attP (phage attachment site) and the bacterial genetic element attB (bacterial attachment site). Xis and Int are responsible for the site-specific recombination event between attP and attB. The recombination event is reversible. The direction depends on the combination of proteins being expressed (see diagram below).

R6K origin and pir: The pir gene encodes the trans-acting protein that allows replication of an R6K origin2. When pir is turned on, a plasmid containing the R6K origin will be allowed to replicate, but when pir is not expressed the plasmid becomes a suicide vector (cannot replicate). The CRIM system takes advantage of this by placing an attP site in the R6K vector. When pir is off, the vector must insert into the chromosome or be lost.

CRIM (Conditional-Replication, Integration, Modular) plasmids utilize these factors and provide the current means of shuttling a construct into and out of a bacterial chromosome1. However, the design of this system requires manual labor in the form of helper plasmid transformations and subsequent selection procedures each time we wish to excise or integrate our construct (see below figure). In our design we rewire the CRIM machinery to place the integration and excision events under chemical inducers to eliminate this manual process. To tie back into the filing cabinet analogy, we are reworking the system to make the process of removing and replacing a file easier, and taking the first step toward allowing this system to be utilized outside of the laboratory by placing the filing system under environmental signals rather than laboratory procedures.

Caption: The CRIM system uses phage machinery placed on helper plasmids to promote integration and excision events. Preparation of competent cells, transformation of the appropriate helper plasmid, and a multi-step selection process must be performed for each integration and excision event. The helper plasmids contain a temperature sensitive origin (oriR101) and may be cured via 37C incubation.

The design of our E. chiver system seeks to obtain the following goals: 1) Link integration, excision, and replication of the construct (file) to the presence or absence of a chemical inducer. 2) Allow for modularity of the chemical inducer and gene of interest (file contents). 3) Ensure energetically favorable storage of construct by integration as a single copy (avoid multiple integration events). 4) Enable the system to report which machinery is being expressed. 5) Ultimately allow for the coexistence of multiple files to create a bacterial filing cabinet.

A single File contains two major components

For our example of a single file design (addressing goals 1-4) we used lambda phage machinery. However, any Lambdoid phage machinery can be by replaced into this system in order to change the site of chromosomal integration. Follow the link below for a walk through of our single file design and an explanation of how it obtains goals 1-4.

Our design addressing goal 5, allowing for multiple files to coexist (a true filing cabinet), was explored by adding an analogous second filing system under the control of P21 phage machinery and the conditional replication of origin OriV to our original Lambda system. The other difference in this P21 construct is the use of the CII repressor to repress the P21 integrase gene when appropriate. In order to create a multi-file system each analogous construct (file) must have 1) a unique origin of replication, 2) a separate Lambdoid phage system whose activity does not overlap with other Lambdoid phage sites (i.e. the combination of HK022 and Lambda phage files is not recommended because HK022 recognizes Lambda attachment sites as well as it’s own), 3) no two files may have their helper construct integrase gene under the same repressor (i.e. lambda system utilizes cI while P21 system utilizes cII). These three requirements are necessary to prevent crosstalk between two files. Click on the link below to view a walk through of the multi-file, Lambda and P21, system.

1. Haldimann, A., Wanner, B. L. 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology. 183:21 6384-6393. 2. Metcalf, W. W., Weihong, J., Wanner, B. L. 1994. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kϒ origin plasmids at different copy numbers. Gene. 138: 1-7.