Help:Protein domains/Construction

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Oftentimes, Registry users don't bother to construct proteins domains as individual parts. Instead, they

  1. Design the individual protein domains in the Registry.
  2. Specify a translational unit as a combination of a Head domain, one or more Internal domains and a Tail domain.
  3. Synthesize the complete translational unit or protein coding sequence via direct commercial gene synthesis.

By using commercial gene synthesis, you can ensure that your translational unit or protein coding sequence doesn't have any scar sequences and is codon-optimized for your chassis of choice.

If, however, you do want to construct individual protein domain parts and then assemble them later, then you have a few different options depending on the length and source of your particular part.

  1. Constructing a part by de novo DNA synthesis: Long, synthetic parts likely need to be constructed via de novo DNA synthesis.
  2. 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.
  3. Constructing a part by PCR: Long parts designed from naturally occurring genetic sequences can instead be amplified via PCR.

Constructing a part by de novo DNA synthesis

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

1. Design your part

  1. Design your protein domain sequence so that the codon usage is optimized for the chassis in which the part will be used.
  2. Make sure that the protein domain sequence doesn't have any BioBrick sites in it (EcoRI, XbaI, SpeI, or PstI). If it does, you'll need to remove them.
  3. Also design your protein domain sequence to remove useful restriction enzyme sites. See a list of suggested sites for removal at OpenWetWare.
  4. Enter the sequence of your part in the Registry as a new part. But don't include any Assembly standard prefix or suffix sequences in the sequence you enter in the Registry!
  5. Add the Protein domain prefix to the 5' end of the part and add the Protein domain suffix to the 3' end of the part.
    • For Head Domains, use the standard BioBrick protein coding sequence prefix 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA G-3' followed an ATG start codon.
    • The sequence of the Protein domain prefix for Internal Domains, Special Internal Domains, and Tail Domains, depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.
    • For Tail Domains, the double TAA TAA stop codon should be followed by the standard BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
    • The sequence of the Protein domain suffix for Head Domains, Internal Domains, and Special Internal Domains depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.

2. Order the DNA sequence

  1. Place an order for your new part with a commercial DNA synthesis company.
  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 Protein domain suffix and ending with the Protein domain prefix. See help on entering new plasmid backbones in the Registry.
  • Also, don't forget to submit your new part to the Registry.

Constructing a part from synthetic oligos

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

Many protein domain parts, particularly Head Domains, Special Internal Domains, and Tail Domains, are relatively short sequences. 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.

1. Designing the oligos needed to make a part

  1. Design your protein domain sequence so that the codon usage is optimized for the chassis in which the part will be used.
  2. Make sure that the protein domain sequence doesn't have any BioBrick sites in it (EcoRI, XbaI, SpeI, or PstI). If it does, you'll need to remove them.
  3. Also design your protein domain sequence to remove useful restriction enzyme sites. See a list of suggested sites for removal at OpenWetWare.
  4. Enter the sequence of your part in the Registry as a new part. But don't include any Assembly standard prefix or suffix sequences in the sequence you enter in the Registry!
  5. Design a forward primer to your new part comprised of the Protein domain prefix sequence followed by at least the first 20 or so nucleotides of the part.
    • For Head Domains, use the standard BioBrick protein coding sequence prefix 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA G-3' followed an ATG start codon.
    • The sequence of the Protein domain prefix for Internal Domains, Special Internal Domains, and Tail Domains, depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.
    • 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.
  6. 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 Protein domain suffix sequence
    • For Tail Domains, the double TAA TAA stop codon should be followed by the standard BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
    • The sequence of the Protein domain suffix for Head Domains, Internal Domains, and Special Internal Domains depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.
  7. 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 Invitrogen or 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 here.
  3. Once your primers are resuspended, you need to anneal and extend them to make the full length double-stranded part via 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. Design your protein domain sequence. Choose the boundaries so that the part can be reused by others as much as possible.
  2. Enter the sequence of your part in the Registry as a new part. But don't include any Assembly standard prefix or suffix sequences in the sequence you enter in the Registry!
  3. Design a forward primer to your new part comprised of the Protein domain prefix sequence followed by the first 20-30 nucleotides of the part.
    • For Head Domains, use the standard BioBrick protein coding sequence prefix 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA G-3' followed an ATG start codon.
    • The sequence of the Protein domain prefix for Internal Domains, Special Internal Domains, and Tail Domains, depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.
    • For more guidelines on how to design the portion of the primer that matches the template sequence, see the help page on designing primers.
  4. Design a reverse primer to your new DNA part comprised of the last 20-30 nucleotides of the DNA part sequence followed by the Protein domain suffix sequence.
    • For Tail Domains, the double TAA TAA stop codon should be followed by the standard BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
    • The sequence of the Protein domain suffix for Head Domains, Internal Domains, and Special Internal Domains depends on which Protein domain assembly standard you choose to use. See Help:Protein domains/Assembly for details.
  5. 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 Invitrogen or 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 here.
  2. Using the resuspended oligo's, do a PCR of the template DNA.
  3. Purify the PCR product using either agarose gel purification (to select full length PCR products) or 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 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 site-directed mutagenesis so that the assembled protein coding sequence conforms to the BioBrick standard and can be used in subsequent assembly steps.