Due to the long coding regions of our constructs that require the assembly of multiple fragments, the
absence of scar between fragments is important. Therefore, we chose Seamless Ligation Cloning Extract
(SLiCE) method for assembly of most of our constructs, which is similar to Gibson assembly where an
overlapping region was added to the ends of the fragment to be ligated. Exonuclease activity in the
bacterial extract will create a sticky end by “chewing back” from the 5’ ends, enabling a seamless
ligation and assembly of multiple fragments at the same time. For some of the constructs, Gibson
assembly was also tried. For more details, please refer to the
SLiCE/Gibson assembly protocol
.
In our project, all cloning works are carried out in E. coli strain DH5α, and then the
plasmids
are
linearized and transformed into Yarrowia lipolytica by the lithium acetate method.
The following chart shows the general cloning workflow carried out throughout our project:
The following part shows the cloning works performed in our project:
pTEFin-LIP2t-pSB1C3
We ordered a popular Yarrowia lipolytica constitutive promoter for Translational Elongation
Factor-1α
with an intron sequence (pTEFin, BBa_K5159005) and terminator LIP2t (BBa_K5159006) from
Integrated Genome
Technology (IDT). The intron sequence is reported to increase the promoter strength by 16 fold, where in
our
project, a strong expression level of the PUFA synthase is desired for detection and synthesis of DHA.
Using SLiCE assembly, we first assembled a new backbone by inserting pTEF and LIP2t into the common E.
coli plasmid pSB1C3 between prefix and suffix. It is successfully cloned and sequenced to ensure the
accuracy. This facilitates the further construction of our Yarrowia lipolytica expression cassettes with
the same promoter and terminator combination.
pTEFin-PPT-LIP2t-pSB1C3 & pTEFin-PPT-LIP2t-LEU2 cassette-pSB1C3
The cassette expressing PPTase under pTEFin was successfully cloned by SLiCE assembly of PPT with the
pTEFin-LIP2t-pSB1C3 linearized with overhang primers. The construct was digestion-checked with
restriction enzymes (BamHI+StyI) and sequenced, ensuring the assembly is correct.
In order to add the LEU2 selection marker to the construct for transforming Yarrowia lipolytica,
we then
digested plasmid pTEFin-PPT-LIP2t-pSB1C3 with SpeI and PstI, and linear LEU2
expression cassette
(BBa_K5159008) with XbaI and PstI. The two digested fragments were then gel
purified, ligated and
transformed into E. coli. However, due to time limitation and unexpected difficulties met during
purification and ligation, despite multiple attempts, we are unable to obtain the ligated final
construct.
Alternatively, we linearized pTEFin-PPT-LIP2t-pSB1C3 by amplifying it with primers VF2 and
VR, and
directly transformed it into Yarrowia lipolytica on a YPD plate without selection. Colony PCRs
were
performed to screen the successfully transformed colonies, however no correct transformant was
identified.
pTEFin-C1-C2-LIP2t-pSB1C3 & pTEFin-B1-B2-B3-LIP2t-pSB1C3
Same approach was applied to the assembly of constructs expressing PUFA synthase subunit B and subunit C.
As ORF_B and ORF_C are divided into 3 and 2 fragments respectively for an easier synthesis purpose
required by IDT, Gibson assembly of 4-piece and 3-piece was performed. However, a very low assembly
efficiency was observed. Different ratios between inserts and the vector were tested, but still no
correct colony was found in cPCR after transformation.
In order to accelerate the cloning process, we also did directly PCR with primers VF2 and
VR and gel
electrophoresis after the Gibson assembly. Via this method the correct band size (5730 bp) corresponding
to the successful assembly of pTEFin-C1-C2-LIP2t-pSB1C3 was observed, however, due to technical
difficulties, we were unable to purify enough correct DNA from the gel for transformation into
Yarrowia
lipolytica.
pTEFin-GFP-LIP2t-pSB1C3
The construct expressing Green Fluorescence Protein (GFP, BBa_E0040) under pTEFin is
assembled for
testing the promoter strength and serves as a standard control for quantifying strength of other
promoters to be tested. It was successfully constructed by SLiCE assembly of GFP and
pTEFin-LIP2t-pSB1C3
linearized with overhang primers. The construct was digestion-checked with restriction enzymes
StyI,
ensuring the assembly is correct.
The construct was then linearized by amplifying it with primers VF2 and VR, and directly
transformed into
Yarrowia lipolytica on a YPD plate without selection, as the addition of LEU2 selection marker to
pTEFin-PPT-LIP2t-pSB1C3 still showed no success. Unfortunately, no colony with distinguishable
fluorescence was observed on the transformed plate.
pCIT1/pIDP2-GFP-LIP2t-pSB1C3
The 2 constructs expressing GFP were cloned to characterize inducible promoters CIT1 and
IDP2 in their
native strain Yarrowia lipolytica. The assemblies were successfully done by SLiCE of the promoter
sequences with overhangs, and the pTEFin-GFP-LIP2t-pSB1C3 linearized with overhang primers
removing
pTEFin. The constructs were digestion-checked with restriction enzymes ApaI+StyI and StyI
respectively,
ensuring the clones are correct.
As the case for pTEFin-GFP-LIP2t-pSB1C3, the 2 cloned constructs were then linearized by PCR and
directly
transformed into Yarrowia lipolytica without selection. However, also no colony with
distinguishable
fluorescence was observed on the transformed plate.
PEX10 3’ region-LEU2 cassette-PEX10 5’ region-pSB1C3
Similar to the assembly of pTEFin-C1-C2-LIP2t-pSB1C3, this time a 4-piece Gibson assembly was
carried out
after amplifying the fragments with overhang primers. However still no correct assembly was observed
after multiple rounds of trials.
