Practical skills in Biochemistry A
Cloning Workshop
Aim
To design and verify plasmid DNA constructs for the expression of the fluorescent Light-Oxygen-Voltage (LOV) domain from the Arabidopsis thaliana phototropin 2 protein in a standard E. coli protein expression vector and a vector modified for Golden Gate cloning.
Before attending this workshop we recommend you acquaint your self with the Polymerase Chain Reaction (PCR) and restriction endonucleases. Chapter 7 of Molecular Cell Biology by Lodish et al, has a good overview of molecular cloning techniques:
http://www.ncbi.nlm.nih.gov/books/NBK21712/
Learning outcomes
At the end of this workshop you should be able to:
⦁ Search DNA sequence databases for genomic DNA sequences encoding protein targets.
⦁ Translate nucleotide sequences for open reading frames into protein sequences using online tools.
⦁ Determine domain boundaries in a polypeptide chain.
⦁ Design protein expression constructs based on predicted domain boundaries
⦁ Select suitable expression vectors for protein targets.
⦁ Identify restriction endonuclease sites for insertion into multiple cloning sites of common expression vectors.
⦁ Understand the difference between Type II and Type IIs restriction endonucleases and how they recognise and cut DNA.
⦁ Design primers to amplify genomic/synthetic DNA with appropriate restriction sites and overhangs for classical restriction/ligation and Golden Gate cloning strategies.
⦁ Design a protocol for cloning targets of interest using both classical and Golden Gate strategies.
⦁ Analyse Sanger sequencing data to determine successful insertion of cloned fragments into expression vectors.
Introduction
Before the advent of mass genome sequencing projects, proteins of biochemical interest had to be purified from their source organism or tissue in a series of time consuming and usually messy/cold experiments. Now, we have genome sequences for more than 10,000 bacterial species and thousands of eukaryotes. We can download and analyse the DNA sequences of almost any gene we could conceive of and design protein expression constructs for these in silico. Native genomic DNA, or cDNA, is available from a number of sources, and can often be bought online with a credit card. It is also possible to take native protein encoding DNA and optimise the codon usage for specific organisms and purchase synthetic DNA, or even entire constructs. The Edinburgh Genome Foundry, run by Professor Rosser and Dr Cai will eventually make it possible to design and produce complete synthetic eukaryotic chromosomes.
In this workshop we are going to design protein expression constructs for the fluorescent LOV domain from the Arabidopsis thaliana phototropin 2 protein. This protein is a useful alternative fluorescent marker to Green Fluorescent Protein (GFP) as it uses a flavin mononucleotide cofactor, rather than the protein-derived cofactor of GFP, and unlike GFP it is fluorescent in anaerobic conditions. We wish to produce this protein for biochemical and biophysical characterisation. To do this we must first generate the plasmids to clone and produce the protein fragments for our study, as is common practice in structural and biochemical studies of proteins.
We will explore the design rationale for protein expression constructs using both a standard restriction/ligation and a Golden Gate cloning method (Engler, Kandzia, & Marillonnet, 2008).
Important notes:
Save intermediate DNA/mRNA/protein sequence files as plain text fasta (https://en.wikipedia.org/wiki/FASTA_format) files using notepad/wordpad.
Keep tabs for each different webserver you use open. You’ll need to switch between them a few times.
Part 1
DNA sequence databases
The Kyoto Encyclopedia of Genes and Genomes: www.kegg.jp
NCBI gene and genome database: www.ncbi.nlm.nih.gov
Department of Energy genome database: http://img.jgi.doe.gov/
Multiple sequence alignment:
Clustal omega: http://www.ebi.ac.uk/Tools/msa/clustalo/
Task:
⦁ Find the mRNA sequence of A. thaliana PHOT2 and save the sequence as a fasta file on your computer.
What is the NCBI reference sequence accession number for this mRNA?
NM_180880.1/ http://www.ncbi.nlm.nih.gov/nuccore/5391441
How does a Eukaryotic mRNA differ from the genomic DNA sequence?
Presence of introns in genomic DNA, 5’ UTR, 3’ poly A.
What are the important features of a fasta file?
Starts with ‘>’ and single line descriptor of file
Then sequence on next line.
Can append multiple sequences in one file as long as each one starts with a ‘>’
Saved as plain text, usually ‘.fas’ or ‘.fasta’ extension.
Part 2
We are primarily interested in the protein-coding region of this mRNA, so we need to translate this from nucleotide sequence amino acid sequence and determine the correct reading frame for translation.
Task:
⦁ Translate the nucleotide sequence into amino acids using the Expasy translation server.
⦁ http://web.expasy.org/translate/
⦁ Paste the mRNA sequence into the box and select ‘includes nucleotide sequence’ from the Output format drop down list.
⦁ Click ‘translate sequence’
Which reading frame gives the correct sequence?
5’ frame 1
⦁ If you click on this reading frame you will see the translated sequence for this reading frame. It has a few possible start/stop sites. Select the longest open reading frame by clicking on the initiating methionine.
Paste the full protein sequence in Fasta format here:
>LOV
MERPRAPPSP LNDAESLSER RSLEIFNPSS GKETHGSTSS SSKPPLDGNN KGSSSKWMEF QDSAKITERT AEWGLSAVKP DSGDDGISFK LSSEVERSKN MSRRSSEEST SSESGAFPRV SQELKTALST LQQTFVVSDA TQPHCPIVYA SSGFFTMTGY SSKEIVGRNC RFLQGPDTDK NEVAKIRDCV KNGKSYCGRL LNYKKDGTPF WNLLTVTPIK DDQGNTIKFI GMQVEVSKYT EGVNDKALRP NGLSKSLIRY DARQKEKALD SITEVVQTIR HRKSQVQESV SNDTMVKPDS STTPTPGRQT RQSDEASKSF RTPGRVSTPT GSKLKSSNNR HEDLLRMEPE ELMLSTEVIG QRDSWDLSDR ERDIRQGIDL ATTLERIEKN FVISDPRLPD NPIIFASDSF LELTEYSREE ILGRNCRFLQ GPETDQATVQ KIRDAIRDQR EITVQLINYT KSGKKFWNLF HLQPMRDQKG ELQYFIGVQL DGSDHVEPLQ NRLSERTEMQ SSKLVKATAT NVDEAVRELP DANTRPEDLW AAHSKPVYPL PHNKESTSWK AIKKIQASGE TVGLHHFKPI KPLGSGDTGS VHLVELKGTG ELYAMKAMEK TMMLNRNKAH RACIEREIIS LLDHPFLPTL YASFQTSTHV CLITDFCPGG ELFALLDRQP MKILTEDSAR FYAAEVVIGL EYLHCLGIVY RDLKPENILL KKDGHIVLAD FDLSFMTTCT PQLIIPAAPS KRRRSKSQPL PTFVAEPSTQ SNSFVGTEEY IAPEIITGAG HTSAIDWWAL GILLYEMLYG RTPFRGKNRQ KTFANILHKD LTFPSSIPVS LVGRQLINTL LNRDPSSRLG SKGGANEIKQ HAFFRGINWP LIRGMSPPPL DAPLSIIEKD PNAKDIKWED DGVLVNSTDLDIDLF
Part 3
We want to clone the LOV domain from this protein, so we need to determine what domains the protein sequence encodes and the domain boundaries.
Task:
⦁ Paste your protein sequence into the ‘protein sequence’ box on the SMART page (⦁ http://smart.embl-heidelberg.de) and check the boxes for ‘outlier homologues’, ‘PFAM domains’, signal peptides’ and ‘internal repeats’.
The server will return a graphical view of the different domains in your protein sequence.
Complete the table below with details of the predicted domains
Name Start End
Low complexity 102 114
PAS 122 191
PAC 197 239
PAS 378 447
PAC 453 495
S_TKc 577 864
⦁ Look at the box with outlier homologues and homologues of known structure. This displays known structures with sequence homology to the predicted domains in the protein.
⦁ Click on the names of the domains to get further information.
Which of the domains are related to LOV proteins of known structure?
PAS1 – 2z6d
PAS2 – 2z6d
The domain boundary predictions given by SMART cover only highly conserved regions of predicted domains. To design robust expression constructs you usually need to include a few amino acids either side of the predicted domain to ensure you have the full protein domain, or you may wish to use structural homologues to guide the design of your construct.
Part 4
Construct design
We wish to clone the protein sequence corresponding to the first LOV domain in the protein.
Task:
⦁ Using the homologues as a reference, identify the first LOV domain and click on the link in the homologue view to see the domain in the protein that matches this. Look at the table and identify the start and end residues for this domain and locate these in your translated nucleotide sequence
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