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Department of Chemistry

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1 Department of Chemistry
Cloning 101: A Primer Ghosh Lab University of Arizona Department of Chemistry

2 Outline Cloning overview pDRAW32 Design Gene Insert Primers
Further considerations (optimization of the process) Transformation

3 Cloning Overview Insert (your gene) + Functional Plasmid construct
Four main steps in cloning: Insert synthesis Restriction enzyme digest Ligation Transformation Insert (your gene) + Functional construct Plasmid (vector)

4 Design Overview Functional construct
Steps to follow in designing your cloning experiment: Design your gene Design your insert Pick your enzymes Check your design Recheck your design Functional construct

5 pDRAW32 Plasmid maps: pDRAW32
All of the important information in one place!

6 pDRAW32 You can look at the sequence in detail Open reading frames
Translation Restriction sites Complementary strand

7 Design of the Gene If you are cloning out of a known plasmid, just use the sequence that you have Example, the gene we want: G C D R A S P Y C G We got this from phage display: ggctgcgacagggcgagcccgtactgcggt G C D R A S P Y C G Phage sequence Final sequence for the gene of interest: ggctgcgacagggcgagcccgtactgcggttaa G C D R A S P Y C G * Add a stop codon

8 Design of the Gene If you are designing the gene from scratch, keep in mind codon usage Not all codons are created equal Un-optimized codons could lead to lower expression levels The codon usage reflects levels of tRNA available in E. Coli Pay attention to the stop codons too (XL1-Blues read through TAG {amber stop codon} 20% of the time)

9 What if we don’t have the DNA sequence?
Design from scratch! (don’t forget about codon usage) or preferably…

10 Choice of Restriction Sites/Enzymes
Once you have your gene, you need to design a way to get it into your plasmid Endonucleases (or restriction enzymes) are enzymes which cut DNA at specific internal recognition sequences Compare to exonucleases, which cut from one end You must choose restriction sites that are available in the plasmid you are cloning into They must not appear in your gene (silent mutation can remove unwanted sites in your designed gene)

11 Really Important Factors to Remember When Choosing Restriction Enzymes
Restriction sites must exist only once in your plasmid They must be in the correct position relative to the purification tag Restrictions sites usually add extra residues to your gene product; make sure they are compatible with your peptide/protein Some restriction sites are sub-optimal for cloning Blunt end sites dam and dcm methylation-affected enzymes

12 Blunt vs Sticky Ends Most common restriction enzymes “sticky ends” + +

13 dam Methylation Dam methylase Dam methylase puts a methyl group on the nitrogen of 6th position of adenosine at the site: GATC All of the E. Coli that we use generate DNA with dam methylation Some enzymes only cut dam methylated DNA: eg DpnI Some enzymes do not cut dam methylated DNA: eg XbaI

14 dcm Methylation Dcm methylase Dcm methylase puts a methyl group on the carbon of 5th position of cytidine at the site: CCAGG and CCTGG The enzyme we use most that can be affected by dcm methylation is SfiI XL1-Blues and BL21s are both Dcm+

15 Design of the Insert Once you have your restriction enzymes chosen, it is time to design the final complete gene The multiple cloning site (or whatever plasmid you are cloning into) should already have the 5’ portion of the gene intact (i.e. RBS, spacer, Met) Sequences must be in frame NcoI BtgI 51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC M G S S H H H H H H SacI AscI SbfI SalI NotI BamHI EcoRI EcoICRI BssHII PstI AccI HindIII 101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC S Q D P N S S S A R L Q V D K L A

16 Design of the Insert Multiple cloning site
71 ATGGGCAGCAGCCATCACCATCATCACCAC M G S S H H H H H H SacI AscI SbfI SalI BamHI EcoRI EcoICRI PstI AccI HindIII 101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A The gene we want: ggctgcgacagggcgagcccgtactgcggttaa G C D R A S P Y C G * Be aware of the amber stop codon: TAG BamHI PstI AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A G C D R A S P Y C G * ggctgcgacagggcgagcccgtactgcggttaa AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

17 Design of the Insert Always check and re-check your sequence!
ATGGGCAGCA GCCATCACCA TCATCACCAC AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA Translate the whole gene atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D Everything looks good: in frame the whole way!

18 Design of the Insert The wrong way to do it:
AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT The gene is just inserted after the restriction site, which is out of frame with the plasmid-encoded start-codon/His-tag atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagcc M G S S H H H H H H S Q D P A A T G R A cgtactgcggttaactgcaggtcgacaagctt R T A V N C R S T S Frame shifted = garbage! **Some plasmids, for whatever reason, have restriction sites out of frame with the translated gene**

19 Finishing Touches Restriction enzymes need 5’ and 3’ base pairs to cut properly NEB has a reference guide for specific enzymes (see link below) A good rule of thumb is 6 base pairs after the recognition site Inserting a GC “clamp” at the end and beginning of the sequence is also a good idea atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D Final gene, polished and ready to go: gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D

20 Design of the Primers + Insert Vector
Once the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategy Three main strategies towards insert synthesis: PCR amplification Klenow extension of overlapping primers Complimentary full-length primers + Insert Vector

21 PCR Amplification of Insert from an Existing Gene
The most common method of insert synthesis Necessitates a pre-existing construct Extra restriction sites and/or amino acid residues can be added on each side of the gene Internal mutations are more difficult Insert

22 PCR Synthesis of Insert
PCR amplification from overlapping primers No pre-existing construct is needed PCR products messy, possibly making subsequent rxns difficult Good for inserts >150 bp F1: 10x 5’ 3’ F2: 1x 5’ 3’ 3’ 5’ R1: 1x 3’ 5’ R2: 10x Insert Full-length insert should still be the major product

23 Klenow Extension of Overlapping Primers
Two primers that are complimentary in their 3’ region are designed (overlap  15bp) Extended to full length by the Klenow fragment of DNA Polymerase I Useful if insert is 50 to 150 bp 5’ 3’ 3’ 5’ Insert 5’ 3’ Klenow Klenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activity

24 Complimentary Full-Length Primers
The simplest approach Order two primers that compliment each other Mix the two primers, heat, and aneal slowly (to ensure proper base-pairing) Feasible if the total insert size is < 60 bp 5’ 3’ Anneal Insert 3’ 5’

25 Designing Primers to Order
Once the insert synthesis technique is decided, primer design is fairly straight-forward Forward primers: Assess necessary overlap and copy the sequence from your designed gene, along with extra 5’ sequence Reverse primers: First, design exactly as if it were a forward primer: Copy necessary overlap and extra 3’ sequence from your designed gene Once all this is in place, use pDRAW32 sequence manipulator to calculate the reverse compliment Order the pDRAW32 calculated sequence directly

26 Cloning Out an Existing Gene
In the example mentioned previously, we would normally use full length overlapping primers, but let’s look at the more common case of having a preexisting gene: Preexisting gene: Overlap tgcggcccagccggccatgggctgcgacagggcgagcccgtactgcggtggaggcggtgctgcagcgc A A Q P A M G C D R A S P Y C G G G G A A A + Goal gene: gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D Extra sequence from gene design gccagccaggatccgggctgcgacagg ccgtactgcggttaactgcaggtcgacgc Forward Primer: Design of Reverse Primer:

27 Ordering Primers & Forward primer to order: Design of Reverse Primer:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D Forward primer to order: gccagccaggatccgggctgcgacagg Design of Reverse Primer: ccgtactgcggttaactgcaggtcgacgc & Reverse primer to order: GCGTCGACCTGCAGTTAACCGCAGTACGG Now we can order the primers:

28 Vectors and Bacteria Strains
An important thing to think about before you start cloning: What vectors/E Coli should I use? Vector Promoter E Coli strains we use pQE-30 T5 promoter XL1-Blue: mostly good for DNA isolation/phage display M15(pREP4): tighter regulation of the lac suppressor pMAL Ptac promoter pCANTAB-5E Plac promoter pET-Duet pRSF-Duet T7 lac promoter (An E. Coli strain with phage T7 RNA polymerase is necessary) BL-21: Protease deficient, stable to toxic proteins, and contains the T7 RNA polymerase gene

29 lac Expression Regulation
RNA polymerase lac site Promoter RBS ATG- your gene X IPTG (or lactose, etc) lac repressor lac site Promoter RBS ATG- your gene IPTG Transcription lac site Promoter RBS ATG- your gene mRNA

30 Purification Tags and Selection (Anti-biotic Resistance)
Anti-biotic resistance (working concentration) Ampicillin (100g/mL) Kanamycin (35g/mL) Tetracycline HCl (10g/mL) Chloramphenicol (170g/mL in ethanol) Purification Tag His-tag (nickel agarose resin) Maltose Binding Protein (amylose resin) Glutathione S-Transferase (glutathione resin)

31 Digestion of Insert and Vector
Digest with the same restriction endonucleases Optional (recommended) step: Treat the plasmid DNA with Antarctic phosphatase Decreases the background by stopping self-ligation of singly cut plasmid and background re-ligation

32 Ligation of the Insert into the Vector
+ Ligation covalently attaches the vector and the insert via a phosphodiester bond (5’phosphate and 3’ hydroxyl of the next base)

33 Antarctic Phosphatase and Ligation
Antarctic Phosphatase cleaves this phosphate, disallowing self-ligation The insert still has the 5’ phosphate though

34 Transformation The functional construct is now ready to be transformed into new E. Coli and grown up The new DNA isolated from the E. Coli must then be sequenced to make sure that everything worked Once the sequence is confirmed, we are ready to go!

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