Summer placement is a good way to gain valuable and hands-on work experience in a research lab.  It will help you to develop your skills and prepare you for your future career.  Placements for the following year are always advertised from November/December period to Late April/early May of the year you will be applying.  It’s advisable you apply in the early months to be confident of securing a place.  Also, try to apply to multiple places because you may not be lucky enough to get a place if you apply only for one placement.  You should also aim at emailing academic staff in the biological research lab at Newcastle University requesting for a placement opportunity over the summer.  Newcastle University also offers vacation studentship opportunities to students in their second year.  If you are finding it difficult to get a placement or don’t know what to do, please speak to a member of the careers service or to your tutor.

PS: This article was written in 2013 and was specifically targeted at Newcastle University Biomedical Sciences students. I was one of the beneficiaries of the British Society for Cell Biology (BSCB) funds. With the funds, I was able to conduct my research on the topic ”Does ribosomal protein RPL10 influence translational recoding driven by 2A peptides?”

Information on the project I undertook is below.


2013 Summer Placement 

This page gives a summary of the summer project I had during the 2013 summer break.

Research Project Topic: ”Does ribosomal protein RPL10 influence translational recoding driven by 2A peptides?”

Research Project Awarded to: Mr Kate, Wisdom Deebeke

Name & Address Of Project Supervisor: Dr Jeremy D. Brown, Institute For Cell & Molecular Biosciences, Newcastle University.

Subject Area: Genetics and Biochemistry

Project start & end dates: 17th June to 26th July, 2013

Length of project: 6 weeks

Funding body: British Society For Cell Biology

Summary of Project: A brief summary of the project is outlined below.  In-depth steps that were taken to design the experiment are provided here.

Many viruses subvert the genetic code to drive non-canonical outcomes of translation. Such events are termed ‘recoding’, and are essential for viral gene expression, and hence replication. This project focuses on one recoding event, termed ‘stop-carry on’, which generates separated proteins from a single gene. Viruses that use stop-carry on recoding include a number that affect human health and/or have significant economic impact (e.g. foot-and-mouth disease). In this project Wisdom Kate will alter, and then test, a specific component of ribosomes (the cell’s protein synthesis factories) that we have reason to believe may influence the stop-carry on translational recoding. Information gleaned from this will be highly informative as to the mechanism of the reaction and may also provide information potentially useful in countering viruses that use stop-carry on as part of their gene expression program.
Protein synthesis does not always adhere to the genetic code. Some mRNA, and occasionally nascent peptide, sequences, promote outcomes differing from that specified by the coding sequence. Such events, termed ‘recoding’, include stop codon readthrough and frameshifting, and are most often encountered in viral gene expression. Another predominantly viral recoding event is ‘Stop-Carry on’. This is driven by short peptides, termed ‘2A’, which prompt the ribosome to miss a peptide bond in the polypeptide. Mechanistically, the reaction is a non-conventional termination with the final (proline) codon of 2A in the ribosomal A site, followed by translation restarting on the same codon.
2A peptides are autonomous, function in all eukaryotic systems tested, and have become a tool of choice for co-expression of proteins. Insight into the 2A reaction may aid development of expression systems and, importantly, provide information useful in countering viruses that use 2A, a number of which are economically important (e.g. FMDV, human viruses linked to respiratory and diarrheal illness and insect viruses implicated in bee colony collapse disorder).
We have developed reporters to monitor 2A activity in yeast. The first relies on accumulation of red pigments in the absence of Ade2p, a key adenine biosynthesis enzyme. With this reporter, ‘2A-ADE2’, accumulation of Ade2p is a direct consequence of 2A activity, yielding white colonies on plates. With decreasing 2A activity, colonies become pigmented, providing a simple, quantifiable readout. The second reporter, comprises GFP with an internal 2A peptide (‘2A-GFP’), and provides a positive, fluorescence output when 2A is inactive.
Since 2A functions from within the ribosomal exit tunnel, the reaction that it promotes is likely to depend on contact(s) between 2A and the tunnel and the peptidyl transferase centre (PTC). Several ribosomal proteins make close contact with the nascent chain and tRNAs. The focus of this proposal is one of these, RPL10. Recent structural data indicate that it closely contacts the P-site. RPL10 is then a candidate factor that may influence the 2A reaction.
The key objective for the work is mutagenesis of the region of RPL10 that contacts the PTC to identify variants that affect the 2A reaction. We have reporters for a variety of other translational recoding events, including stop codon read through and frame-shifting. A subsidiary aim of the project will be to determine whether any mutants that we isolate in RPL10 also affect the efficiency of these reactions – i.e. do the mutations specifically affect 2A or do they generally impact on translation and its accuracy.
A relatively short region of RPL10 extends into the PTC of the ribosome. We will use a simple, established method for PCR-based mutagenesis followed by in vivo recombination, to generate a library of mutations in the region of RPL10 coding for this domain of the protein in a plasmid-borne copy of RPL10. This will be done in yeast containing the 2A-ADE2 reporter and in which expression of endogenous RPL10 is repressible (available in the laboratory), allowing phenotypes of the mutant proteins to be revealed and examined. Any mutant rpl10-containing plasmids that bestow low 2A activity will be recovered from colonies and re-transformed into the same yeast strain, but containing 2A-GFP, to confirm the phenotype. To examine the effects of rpl10 mutants on stop codon readthrough or frame-shifting, reporters of these events will be transformed into yeast expressing the mutant RPL10 and assayed.
The project will be supported by laboratory funds, and all reagents, strains, plasmids etc. necessary for the project are already in place within the group.
This project is still being continued, and therefore, no conclusion has been reached yet
  • P.Sharma, et al (2012) 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucleic Acids Res. 40: 3143-51.
  • V.A.Doronina, et al (2008) Site-specific release of nascent chains from ribosomes at a sense codon. Mol. Cell. Biol. 28: 4227-39.
Coming soon

Plan of Project: 

The project plan was carried out on a weekly basis as outlined below, although the plans were not strictly adhered to – due to reasons of experiment not properly working as expected, or that I had to repeat a particular step over days.

Transform 2A-ADE2 reporter plasmid into RPL10 on/off strain. Trial in vivo recombination protocol for RPL10 in RPL10 on/off strain
Large scale transformation of conditional RPL10 strain to generate rpl10 mutant library. Pick pigmented colonies from screening plates, restreaking to confirm phenotype.
Recovery of plasmids containing mutant RPL10 and sequencing. Retransformation of the plasmids into strains to test with 2A-GFP.
Testing any mutants isolated for effects on other translational recoding events.
Complete laboratory work, write up report, prepare poster.