We are very excited today to announce the publication of the high-throughput knock-out screen on asexual stages using the PlasmoGEM resource. Our study revealed a much higher than expected rate of essentiality of genes for normal blood-stage growth. The paper is available from Cell here.Please also visit the Sanger Institute Website for a news item, describing our work.
We have simplified the request procedure for repeated requests. From now on, an MTA needs to be set up only once and repeated requests can be made under that MTA.
If you have an older MTA with us (predating November 2016) you will need to set up one more MTA with us at your next request. An MTA is valid for one research group as long as it remains at the host institute the MTA was set up with.
For more details, please see our request information pages.
We are pleased to announce a preliminary beta release of blood-stage fitness data from barcode-sequencing experiments with more than 2,500 PlasmoGEM vectors.
Please use the "Phenotype data" link in the main menu to access the data.
We have started to produce PlasmoGEM vectors for P. chabaudi. The vector designs are now available to browse and download through the web interface. Finished vectors will be added as they become available.
It is now possible to search PlasmoGEM with more than one gene identifier at once. In addition, the results of such a search can now be downloaded as a spreadsheet.
We have updated the vector and library clone request forms. Please use the new versions for new requests:
A new version of the P. berghei genome has been released on GeneDB. This updates the underlying genomic sequence as well as many gene models. One consequence is that genes are now referred to by an updated set of gene identifiers. We have updated our links to GeneDB so that requests are redirected to the gene model with the new, version 3, gene identifier. This approach uses the GeneDB search functionality, resulting in a slightly increased response time. We hope this is not causing too much inconvenience. We will update PlasmoGEM to the new genome but this is likely to take some time since this is a major update.
We have recently generated a near genome-scale P. berghei artificial chromosome resource. Large (6-9 kb) inserts of P. berghei genomic DNA are maintained in a custom version of the Lucigen OC blunt pJAZZ vector, which has been equipped with a P. berghei centromere and flanking telomers to facilitate the stable inheritance of the artificial chromosome upon transfection of P. berghei. This resource can be used to complement any gene knock-out phenotype for which there is an artificial chromosome clone available covering the corresponding gene (publication in preparation). To search the resource, tick 'artificial chromosomes' in the 'Genes' search box to filter results for genes that are fully covered by PbAC clones.
This manuscript describes the PlasmoGEM vector resource and its use in high-throughput reverse genetic screening. The paper shows how the resource can be used for phenotyping dozens of mutants in a single mouse by utilising the barcode sequences that are part of our standard vectors.
Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail M, Herd C, Pfander C, Modrzynska K, Rayner JC and Billker O: A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite. Cell Host & Microbe(17), 404-413, DOI: http://dx.doi.org/10.1016/j.chom.2015.01.014
The PlasmoGEM software and web portal manuscript has now been published in the current database issue of Nucleic acids research: http://nar.oxfordjournals.org/content/43/D1/D1176.abstract In addition, a manuscript describing the biological resource and how it can be used for high-throughput reverse genetic screening has been accepted by Cell Host & Microbe and will become publicly available soon.
The construct and quality control feature browsers that show up on design and quality control details pages will now display the actual DNA sequence at high zoom levels.
Constructs made for the PlasmoGEM resource are quality controlled by deep sequencing. By default, we produce 4 clones for each new construct, which are subjected to deep sequencing and automatically analysed for mutations. The results of the quality control sequencing analyses are now publicly available on our website. To view the data, follow the link given in the Available clones implementing design field on the details page for a design. For example here. Where mutations have been detected, a feature browser window is displayed to investigate the mutations in the context of the affected design, e.g. here. A genbank file with all annotations including the detected mutations can also be downloaded. We distinguish between critical and non-critical mutations. More details are available here.
The search and browse functionality has become more intuitive: Text or gene ID searches now return gene lists with buttons that indicate immediately which type of resource is available, i. e. genomic library clones, tagging and/or knock out vectors. If no button appears, the resource has not been produced. Searches can be limited to genes with available tagging and/or knock out vectors. Clicking through from the search result to the final vector leads to a page that also displays intermediate vectors. Designs are now listed by date. The latest design, usually implementing the most advanced design criteria, is highlighted. From pages showing only knock-out or tagging designs, a link now leads to a page to browse all reagents we have for a given gene.
Updated the Material Transfer Agreement as well as PlasmoGEM protocols and vector information documents.
For those PlasmoGEM users that are making their own vectors from scratch or Zeo/PheS intermediate vectors, please note that we have now switched from zeocin single selection, to zeocin and kanamycin double selection following the recombineering step of our vector production protocol. This means that we are also growing all our Zeo/PheS intermediate vectors in zeocin and kanamycin at all times, and we recommend that you do too. Continuous culturing in zeocin alone can lead to Zeo/PheS intermediate vectors losing the part of the vector encoding for kanamycin resistance.
see news item from 12/07/2013 for details
Fixed an error that had dissociated the barcode from the R2 end of the selection cassette in some knock out designs displayed on the website. No finished constructs were affected. PlasmoGEM vectors always have the barcode on the R2 end of the selection cassette. Whether the barcode is encoded by the downstream or the upstream recombineering oligo (recDown or recUp, respectively) may vary. Vector maps for new designs now show the two universal annealing sites for barcode amplification as “BA-primer” and “BA_2”.
We have recently made a change to our quality control (QC) methods for PlasmoGEM vectors, in order to fulfill our goal of only releasing vectors of the highest possible quality. Previously, PlasmoGEM vectors were tested using a combination of PCR and NotI restriction digest QC steps. During the last few months we have started to sequence the full genomic DNA insert of all PlasmoGEM vectors in large batches using Illumina technology. With this we can now compare the complete sequence of each vector with the expected nucleotide sequence, which allows us to detect the vast majority of potential problems. We now use this as our primary QC for all new vectors. We are also retrospectively sequencing the entire PlasmoGEM resource. From now on we will only release constructs and display them on the PlasmoGEM database when they pass this new level of sequence QC. This does mean there will be a minor delay until new vectors appear on the database. A small number of vectors have failed QC and will be removed from the resource. Please note that sequence errors in extremely AT-rich and low complexity regions are difficult to identify reliably. Also, if we cannot provide a sequence perfect vector, we will tolerate SNPs and small indels in non-coding regions. Additional information is available here, outlining the criteria we use to make the QC sequencing pass and fail calls. Eventually we will make the actual QC data available through the database, so you can judge the quality of each vector yourself.
Improvements in the rules governing vector design (see recent news items) have led to new, alternative, designs being added for some genes. Designs for which we have shipped vectors already will remain in the database.
Please note that in all new deletion vectors we will now reverse the orientation of the selection cassette relative to the direction of the target gene.Where the KO design leaves a short 5' fragment of the target gene, the old orientation increased the risk of a short transcript being stabilised and expressed. This is due to the 3' utr of the triple HA tag that is included in the selection cassette. We are now pointing this element in the opposite direction. We are remaking the small number of vectors where the old orientation is likely to cause a problem. For existing vectors the old design will remain on the database, but those KO designs that we have not yet turned into vectors have changed to the new orientation. Please note that the orientation of the selection cassette has an impact on the primer to use in conjunction with the GT genotyping primer. Which primer combination to use is shown in the details view for each transfection vector.
Based on your feed-back we have made a change to our shipping arrangements. The requestor is still responsible for paying the shipping fee, but the shipment itself is arranged by the PlasmoGEM team. In order to facilitate this we will ask for your shipping account details for your preferred company and your delivery address. When the shipment has been made, we will provide you with the tracking number so you can track your delivery.
We have added a field under the quality control section in the transfection vector details page to indicate which combination of oligonucleotides you should use for the genotyping PCR.
For those of you who produce your own vectors from scratch or produce your own transfection vectors, we have made improvements to the Gateway component of our protocol. An updated version of the protocol is available here.