Biomedical Engineering Reference
In-Depth Information
Fig.
7
Freiburg assembly prefix and suffix sequences. The original RFC 10 prefix and suffix sequences are
underlined
replacing TCn with either AGT or AGC, as all these codons would
encode for the same amino acid, serine.
For parts to be compatible with the Silver standard, it is impor-
tant that they start and end in frame, and that no spacer nucleotides
are present, as these would alter the reading frame of the protein.
RFC 25, called the Freiburg standard, is another assembly standard
designed to facilitate protein fusion by shortening the spacer or
“scar” sequence between parts [
8
]. It was proposed by the Freiburg
iGEM 2007 team; it is backwards compatible with RFC 10 and is
intended as an alternative to RFC 23. RFC 25 extends RFC 10 by
allowing modular construction of fusion proteins by the use of
BioBrick “FusionParts,” defined in the standard (Fig.
7
).
The underlined part of the sequence in Fig.
7
is the original RFC
10 prefix and suffix. It is important to note that the FusionParts pre-
fix contains a start codon directly after the end of the RFC 10 prefix
sequence. As a result, if a FusionPart is made from a complete
protein-coding sequence, the amino acid sequence methionine-
alanine-glycine will be added to the start (N-terminus) of the protein.
This can be avoided by using a hybrid “N-part” format, which is
done by using the RFC 10 prefix in place of the RFC 25 prefix.
FusionParts can be used as standard BioBrick Parts according
to RFC 10. The scar sequence is longer than for RFC 10, as it
contains the additional restriction sites AgeI or NgoMIV. Since the
RFC 25 prefix and suffix contains the RFC 10 prefix and suffix,
assembly can be performed like described in Fig.
1
. Alternatively,
the AgeI and NgoMIV sites can be used for assembly of fusion
proteins, as depicted in Fig.
8
. The AgeI and NgoMIV overhangs
are compatible, with ligation resulting in the shortened scar
sequence ACCGGC, coding threonine and glycine. As for the
RFC 12, 21, and 23 standards, this scar contains neither a frame-
shift nor a stop codon. Compared to RFC 23, RFC 25 avoids
potentially destabilizing changes to the N-terminal of the protein,
and native protein start can be kept intact by using N-parts.
RFC 25 is compatible with Standard Assembly, 3A Assembly,
and Gibson Scarless Assembly (see below), using the established
protocols for RFC 10. With respect to fusion protein assembly,
1.1.5
Freiburg Assembly
