Team:Johns Hopkins-Wetware/yeastgoldengate


JHU iGEM 2012

Yeast Golden Gate (RFC88) describes a new standard for the assembly of basic Saccharomyces cerevisiae transcriptional units (TUs) consisting of a promoter/5'untranslated region (UTR), open reading frame (ORF), and 3'UTR/terminator. Here, "promoter" refers to both the promoter and the 5' UTR, which we currently define as a single part. Future iterations of this standard will incorporate subdivision of currently defined parts e.g. into promoter and 5' UTR. The standard makes use of the type IIS restriction enzyme BsaI to generate standardized and user-defined 'signature overhangs', thus enabling directional and seamless TU assembly. RFC88 is supported by the Yeast Standardized Collection of Parts for Expression (SCoPE), a repository of subcloned and sequence verified parts compatible with this assembly standard. The Yeast SCoPE is housed at Johns Hopkins University and is currently populated by a large number of S. cerevisiae promoters and terminators that facilitate expression and characterization of non-native ORFs.

Click for RFC88

BsaI Cutting
The recognition site of BsaI (red/orange text) is offset from its cleavage site at positions +1 and +5 (white text). A part flanked by BsaI sites oriented in this manner will be left with user defined, 5' 'signature overhangs' (turquoise) following BsaI digestion.

RFC88 utilizes the Type IIS restriction enzyme BsaI, which has a six base pair non-palindromic recognition sequence and cuts at positions +1/+5. Because the enzyme cleavage site is distal to its recognition sequence, the overhang may be user-specified during the design stage. Importantly, if oriented correctly the six base pair BsaI recognition sequence can effectively be eliminated during digestion and ligation, thus allowing two parts with complementary overhangs to assemble without an intervening restriction enzyme 'scar'. Note that in the instance that a part must contain an internal BsaI site, it is possible to substitute the enzyme BsmBI, which generates overhangs with exactly the same characteristics.

Signature Overhangs
Signature Overhangs, TU assembly
Signature overhangs and the directional assembly of a transcriptional unit in a one-pot digestion-ligation.

To enable directional assembly of a TU, we have assigned non-palindromic, 'signature overhangs' to each of the three types of parts. The 5' and 3' ends of all promoters encode the signature overhangs 5'-CAGT-3' and 5'-AATG-3', respectively. Similarly, ORF signature overhangs are designated 5'-AATG-3' and 5'-TGAG-3' and terminators 5'-TGAG-3' and 5'-TTTT-3'. All acceptor vectors encode the overhangs 5'-CAGT-3' and 5'-TTTT-3'. Note that all overhang sequences are given as top strand sequences for clarity. Signature overhang sequences are flanked by BsaI sites oriented so as to eliminate themselves during the digestion reaction and expose the required complementary sticky ends between promoter/ORF and ORF/terminator, and between acceptor vector/promoter and terminator/acceptor vector. Further, for virtually seamless assembly of the TU, start and stop codons are encoded within the signature overhangs that assemble on either side of the ORF.

One-Pot Assembly
One pot assembly of TU
Yeast Golden Gate One-pot assembly of TU

The assembly of a TU may be carried out by simultaneous digestion and ligation, referred to here as a one-pot reaction. Donor constructs, encoding the promoter, ORF, and terminator parts, are introduced into a single reaction vessel along with BsaI and T4 DNA ligase. Further included is an acceptor vector specifically designed to encode signature overhangs, exposed by BsaI digestion, that are compatible with the 5' and 3' ends of the promoter and terminator, respectively. The combination of digestion and ligation leads to essentially reversible digestion of BsaI sites except when components of the TU/acceptor vector ligate; in this instance the BsaI site is eliminated.

Over time the reaction generates more and more of the desired product. We eliminate background of intact donor molecules entirely by (i) a 5 minute at 50&degC BsaI digestion to linearize residual acceptor vector molecules and (ii) encoding a different drug resistance marker in the acceptor vector. Finally, the parent acceptor vector encodes RFP between the BsaI sites. The RFP gene in the acceptor vector yields red colonies such that assembly of the TU in place of RFP is readily identified visually. Successful ligation will result in white colonies whereas re-ligated acceptor vectors will retain the RFP and present as red colonies. In practice, due to the 5 minute 50&degC step that follows the 37&degC incubation, a condition in which BsaI is active and the ligase is not, the background of red colonies is driven below the limit of detection in most instances. Other easily screenable or visual markers may be substituted for RFP and similarly, other combinations of drug markers could be used instead of Kan and Amp.

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