Help:Terminators/Construction

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Depending on the length and source of your particular part, you have a few different options for how to construct a new part.

  1. Constructing a part from synthetic oligos: Many parts are relatively short sequences, such as promoters, RBS's, terminators and many DNA parts. That means that it is often cheaper and faster to order the DNA encoding a part to be chemically synthesized rather than attempting to amplify the part sequence via PCR. This tutorial briefly outlines the steps involved in making a part from synthetic oligos.
  2. Constructing a part by PCR: Long parts designed from naturally occurring genetic sequences can instead be amplified via PCR.
  3. Constructing a part by de novo DNA synthesis: Long, synthetic parts likely need to be constructed via de novo DNA synthesis.

Constructing a part from synthetic oligos

1. Design your part part oligos BCArrow.png 2. Order oligos BCArrow.png 3. Double-stranding

Many parts are relatively short sequences, such as promoters, RBS's, terminators and many DNA parts. That means that it is often cheaper and faster to order the DNA encoding a part to be chemically synthesized rather than attempting to amplify the part sequence via PCR. This tutorial briefly outlines the steps involved in making a part from synthetic oligos. This approach should work for parts up to ~80 bp in length (excluding the BioBrick prefix and suffix).

1. Designing the oligos needed to make a part

  1. Enter the sequence of your part in the Registry as a new part.
  2. Design a forward primer to your new part comprised of the BioBrick prefix sequence 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' followed by at least the first 20 or so nucleotides of the part.
    • You can find a good tutorial explaining where these prefix and suffix sequences come from [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here].
    • For more guidelines on how to design the portion of the primer that matches the template part sequence, see the help page on designing primers.
  3. Design a reverse primer to your new part comprised of at least the last 20 or so nucleotides of the part sequence followed by the BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
  4. Then take the reverse complement of your reverse primer.

Your forward and reverse primers must overlap by >20 bp.

2. Ordering your oligos

  1. You can order your forward and reverse oligos from companies such as [http://www.invitrogen.com Invitrogen] or [http://www.idtdna.com IDT].
  2. These services are cheap and if you order early enough in the day, you can often get your DNA by the next day.

3. Constructing a double-stranded part

  1. Once your single-stranded oligos have arrived, you'll need to anneal them to make a double-stranded part ready for use in an assembly
  2. First, you'll need to resuspend your oligo's since they typically arrive as dehydrated DNA. You can read a protocol for resuspending oligos that is recommended by Invitrogen [http://openwetware.org/wiki/Reconstituting_primers here].
  3. Once your primers are resuspended, you need to anneal and extend them to make the full length double-stranded part via [http://openwetware.org/wiki/Annealing_and_primer_extension primer annealing and extension].
  4. Once you've constructed the full-length double-stranded part, you can digest it just like you would a plasmid. The digested part can be used in any BioBrick assembly.
  5. Don't forget to submit your new part to the Registry.

Constructing a part by PCR

1. Design your part part oligos BCArrow.png 2. Order oligos BCArrow.png 3. PCR amplify

1. Design the oligos need to amplify your part

  1. Enter the sequence of your part in the Registry as a new part.
  2. Design a forward primer to your new part comprised of the BioBrick prefix sequence 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' followed by the first 20-30 nucleotides of the part.
    • You can find a good tutorial explaining where these prefix and suffix sequences come from [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here].
    • For more guidelines on how to design the portion of the primer that matches the template sequence, see the help page on designing primers.
  3. Design a reverse primer to your new DNA part comprised of the last 20-30 nucleotides of the DNA part sequence followed by the BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
  4. Then take the reverse complement of your reverse primer.

2. Ordering your oligos

  1. You can order your forward and reverse oligos from companies such as [http://www.invitrogen.com Invitrogen] or [http://www.idtdna.com IDT].
  2. These services are cheap and if you order early enough in the day, you can often get your DNA by the next day.

3. Amplify the DNA part by PCR

  1. First, you'll need to resuspend your oligo's since they typically arrive as dehydrated DNA. You can read a protocol for resuspending oligos that is recommended by Invitrogen [http://openwetware.org/wiki/Reconstituting_primers here].
  2. Using the resuspended oligo's, do a [http://openwetware.org/wiki/PCR PCR] of the template DNA.
  3. Purify the PCR product using either agarose gel purification (to select full length PCR products) or [http://openwetware.org/wiki/Purification_of_DNA another method of DNA purification].
    • DNA purification is important to eliminate the DNA polymerase prior to restriction digest.
  4. Digest the purified PCR product. The digested part can be used in any BioBrick assembly or cloned directly into a BioBrick vector.
  5. Don't forget to submit your new part to the Registry.

If you're having trouble cloning the digested PCR product, you can instead directly [http://openwetware.org/wiki/Knight:TOPO_TA_cloning TOPO clone] your PCR product into a TOPO TA vector. Then you can digest the BioBrick part from the TOPO TA vector and either move it to a BioBrick vector or use it in a BioBrick assembly.

If your part sequence has any BioBrick sites in it (EcoRI, XbaI, SpeI, or PstI), you'll need to mutate the site(s) out using [http://openwetware.org/wiki/Site-directed_mutagenesis site-directed mutagenesis] to conform to the BioBrick standard and to use the part in subsequent assembly steps.

Constructing a part by de novo DNA synthesis

1. Design your part BCArrow.png 2. Order the DNA sequence

1. Design your part

  1. Enter the sequence of your part in the Registry as a new part.
  2. Make sure that the part sequence doesn't have any BioBrick sites in it (EcoRI, XbaI, SpeI, or PstI). If it does, you'll need to remove them.
    • Short part sequences often don't have BioBrick sites.
  3. Add the BioBrick prefix to the 5' end of the part 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' and add the BioBrick suffix to the 3' end of the part 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.

2. Order the DNA sequence

  1. Place an order for your new part with a commercial DNA synthesis company.
    • GENEART offers [http://www.geneart.com/index.php?id=284 discounted DNA synthesis to iGEM teams].
    • [http://www.idtdna.com/catalog/CustomGeneSyn/Page1.aspx IDT] offers synthesis of DNA fragments less than 500 bp for quite cheap.
    • You can also try DNA2.0.
  2. As long as you included the BioBrick prefix and suffix as in the sequence for synthesis, you should be ready to proceed with your BioBrick assembly.

Remember

  • After you receive the synthesized DNA from the company, make sure that the plasmid backbone sequence has already been entered in the Registry. You can figure out whether the plasmid backbone sequence has already been entered in the Registry by either browsing some preexisting plasmid backbones from DNA synthesis or doing a BLAST search. If the backbone hasn't already been entered, then enter the plasmid backbone as a new part in the Registry. Plasmid backbones should be entered beginning with the BioBrick suffix and ending with the BioBrick prefix. See help on entering new plasmid backbones in the Registry.
  • Also, don't forget to submit your new part to the Registry.