Constructs

A series of tried and tested pBRACT vectors have been designed and tailored for each of the crop species.  These are available to researchers under MTA at a base charge of £20/ construct (minimum order £40) to cover administrative and replenishment costs. 

Available constructs

The pBRACT vectors are based on pGreen, which is a small, versatile vector designed for easy manipulation in E.coli with a high copy number.

To enable the small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is separated into its’ two distinct functions. The replication origin (ori) is present on pGreen, and the trans-acting replicase gene (RepA) is present on an additional vector, named pSoup. Both vectors are required in Agrobacterium for pGreen (or pBRACT) to replicate.

 

The pBRACT vectors have been designed as Gateway™ destination vectors. They therefore use Gateway™ cloning as the method for gene introduction, which is simple and reliable.

To utilise the Gateway ™ recombination reactions your gene of interest first needs to be cloned into a pENTRY vector. Your gene can then be introduced into any one of the pBRACT destination vectors using a one tube reaction.

 

Cereal constructs (suitable for Barley and Wheat)

Name

Function

Description

VectorNTI file

Word file

LB

RB
pBract202 - 35S Hygint - + +
pBract203 - 35S Hygint Gatecass + +
pBract208 - 35S Hygint Gatecass + +
pBract209 - 35S Hygint Gatecass-r + +
pBract204 control 35S Hygint Ubi GUSint + +
pBract207 RNAi silencing 35S Hygint Ubi RNAi + +
pBract210 control 35S Hygint Ubi LUCint + +
pBract211 over-expression without Gateway 35S Hygint Ubi-nos + +
pBract214 over-expression with Gateway 35S Hygint Ubi-nos + +
pBract001 clean gene - Ubi GUSint + +
pSoup20B clean gene 35S Hygint - + +

Brassica constructs

Name

Function

Description

VectorNTI file

Word file

35S Kan

35S Gatecass

pBract102 - 35S Kan - + +
pBract103 - 35S Kan Gatecass + +
pBract507 RNAi silencing nos Kan 35S RNAi + +
pBract114 over-expression +
pBract104 GUS reporter 35S Kan 35S
Gus-Int		 +

Further details on the Brassica constructs

The BRACT transformation protocol for Brassica is based on kanamycin selection, the Brassica series of vectors therefore all carry the selectable marker gene nptII (kanamycin resistance gene).

We have two basic vectors available, one suitable for traditional cloning approaches (pBract102) and one designed for Gateway™ cloning (pBract103).  Both contain 35S::nptII.  Researchers should use these vectors when they want to introduce their own ‘promoter::gene-coding-sequence::terminator’ of choice.

At the request of users we have also produced an over-expression vector (pBract114) containing a 35S promoter and terminator, allowing researchers to simply drop in the coding sequence for their gene of interest.

Gene silencing vector (pBRACT507) a short 300-400bp appropriate fragment from the gene of interest to be silenced is introduced in both directions using Gateway™ cloning. This vector contains the nos promoter to drive the nptII gene.  This is a weaker promoter than 35S and as such selection levels should be reduced in the tissue culture media. For genotype AG DH1012 we suggest lowering the kanamycin levels from 15mg/l to 5mg/l. 

GUS reporter vector (pBract104): This vector is ideal for use when establishing the Brassica protocol within your own laboratory and/or for evaluating and tailoring protocols for other genotypes of interest.  It contains the Gus reporter gene driven by the 35S promoter.

pBract104

GUS expression in callus

Evaluating a genotype's susceptibility to Agrobacterium:
Gus staining of B. rapa callus formed at the base of
cotyledonary petiole explants 3 weeks after inoculation.