Help:Translational units/Construction

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You have two different options for how to construct a new translational unit.

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

Or alternatively, you can assemble your translational unit from available protein domain parts.

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 coding sequence so that the codon usage is optimized for the chassis in which the part will be used.
  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.
  3. Also design your protein coding sequence to remove useful restriction enzyme sites. See a list of suggested sites for removal at [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Constructing_a_BioBrick_part_via_direct_synthesis OpenWetWare].
  4. Enter the sequence of your part in the Registry as a new part.
  5. Before ordering, add the BioBrick prefix 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' followed the RBS sequence and then the ATG start codon.
  6. Most protein coding regions end with a double TAA TAA stop codon sequence followed by the BioBrick suffix 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'.
    • Note that you should NOT enter the BioBrick prefix or suffix sequences to the part sequence in the Registry. These sequences will get added automatically by the Registry software.

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 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.

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 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' followed the RBS sequence, the ATG start codon, and then the first 20-30 nucleotides of the part.
  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'. Most translational units end with a double TAA TAA stop codon sequence followed by the BioBrick suffix.
    • 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.
    • Note that the BioBrick prefix for protein coding regions beginning with an ATG start codon listed above is different from regular BioBrick parts. This altered prefix sequence ensures the proper spacing between the ribosome binding site and start codon in E. coli parts.
    • Note that you should not enter the BioBrick prefix or suffix sequences to the part sequence in the Registry. These sequences will get added automatically by the Registry software.
  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.