The yeGFP can express green fluorescent protein, which can be used as a reporter gene in the process of characterizing the strength of promoters. We optimized the codon sequence of yeGFP to characterize the strength of promoters.
Tsyn7 is a terminator used in S. bombicola to quantify the level of expression or construct the plasmid and device.
Pgki is the promoter from S. bombicola.
Peno is the promoter of enolase from S. bombicola.
Ptef1 is the promoter of the transcription elongation factor 1 from S. bombicola.
Because of the high molecular weight of Cas9 nuclease which cannot freely shuttle through the nuclear pores, we have to add a nuclear localization sequence (NLS) to guide it through the nucleus.
PXA1 is a key gene in the β-oxidation process, affects the S. bombicola using the oleic acid to produce sophorolipids.
SBLE plays a catalytic role in the production pathway of sophorolipid, which changes the configuration of sophorolipid.
UGTB, the gene of glucosyltransferase in S. bombicola, is responsible for the second glycosylation step, verified by constructing UGTB gene deletion strain and measuring extracellular enzyme solution.
It is a period of gene that expresses the resistance of hygromycin.
Rec will express serine recombinant enzyme. According to the β-Rec/six self-excising system, we use this system to recycle hygromycin resistance gene (hph BBa_K3402012).
BBa_K3402050: report circuit
This device is composed of promoter Ptef1(BBa_K3402007), reporter gene yeGFP(BBa_K3402000) and terminator Tsyn7(BBa_K3402001)
The green fluorescent protein (yeGFP) gene can be used as reporter gene and express in Starmerella bombocola. We conducted this device to test if the yeGFP can express in Starmerella bombocola, so we connected this device. Besides, the hygromycin resistance gene (hph) was built into the plasmid as the marker gene to determine if the transformation is successful. Two homologous arms are used to intsert this gene into PXA1 site.
As a result, the yeGFP was succefully expressed in Starmerella bombocola. Finally, we can link different promoters to yeGFP to know the strength of promoters by testing different fluorescence intensity and transcription levels analysis of different strains.
BBa_K3402051: yeGFP expression cassette with NLS
This device is composed of promoter Ptef1(BBa_K3402007), reporter gene yeGFP(BBa_K3402001)
We hope our CRISPR/Cas9 system can work in Starmerella bombicola and give play to its high efficiency. Firstly, we constructed a yeGFP expression device, we add nuclear localization sequence (NLS) to the 3’ ending of yeGFP gene and use Ptef1 as the promoter to express GFP. Besides, the hygromycin resistance gene was also built into the plasmid as the marker gene to determine if the transformation is successful.
We set two groups, control group and experimental group, to observe if there is green fluorescence in the nucleus to know if it will work as we suspected. For the control group, the green fluorescence is diffused in the cytoplasm. For the experimental group, we observed the green fluorescence in the nucleus, then this sequence could be used for the subsequent construction of the CRISPR/Cas9 system.
BBa_K3402053: Self-excising hygromycin marker cassette
This device is composed of upSBLE(BBa_K3402030), Six site(BBa_K3402032), Pgalk(BBa_K3402033),Rec(BBa_K3402034), Tgki(BBa_K3402019), hph(BBa_K3402012), Six site(BBa_K3402032), doSBLE(BBa_K3402031).
We designed the self-excising hygromycin marker cassette which can be used to excise hygromycin resistance gene after screening. upSBLE and doSBLE are homologous arms which are used to construct device by homology directed repair. Pgalk is a galactose-induced promoter, which can control the expression of serine recombinant enzyme (Rec). According to the β-Rec/six self-excising system, serine recombinant enzyme will recognize two adjacent six sites in the same DNA strand and specifically cut off the fragments between two adjacent six sequences.
As a result, the resistance gene can be recycled and the engineered yeast will never show hygromycin resistance.
BBa_K3402054: Over-expression SBLE cassette
This device is composed of upSBLE(BBa_K3402030), Six site(BBa_K3402032), Pgalk(BBa_K3402033), Rec(BBa_K3402034), Tgki(BBa_K3402019), hph(BBa_K3402012), Ptef1(BBa_K3402007), SBLE(BBa_K3402010), Tsyn7(BBa_K3402001), doSBLE(BBa_K3402031).
upSBLE and doSBLE are homologous arms, which means this device will edit the SBLE site. The β-Rec/six self-excising system will help to recycle the hygromycin resistance gene. SBLE is the key gene in changing the configuration of sophorolipid from acid type to lactone type.
When we add this fragment to the Cas9 expression device, we will use promoters with different expression strength as we characterized before to control the expression level of UGTB. Then we can produce different amount of lactone-type sophorolipids.
When the over-expression of SBLE and UGTB both work in the cell, there will be two type of sophorolipids produced by our engineering yeast, acid type and lactone type. Different types of sophorolipids have different functions. The combination of lactone type and acid type may increase the effect of sophorolipid. So, we can control the ratio of lactone and acid type sophorolipids.
BBa_K3402055: Over-expression UGTB cassette
This device is composed of 50bp-upPXA1 (BBa_K3402037), Six site (BBa_K3402032), Pgalk (BBa_K3402033), Rec (BBa_K3402034), Tgki (BBa_K3402019), hph (BBa_K3402012), Ptef1(BBa_K3402007), UGTB(BBa_K3402011), Tsyn7(BBa_K3402001), 50bp-doPXA1(BBa_K3402038).
upPXA1 and doPXA1 are homologous arms, which means this device will edit the PXA1 site. The β-Rec/six self-excising system will help to excise the hygromycin resistance gene. UDP-glucosyltransferase B (UGTB) is the key gene in the synthesis of sophorolipids.
Choose PXA1 as the gene editing site. Three strong promoters Ptef1, Peno and Pgki were knocked in PXA1 site to over-express UDP-glucosyltransferase B (UGTB).
Then we transformed the corresponding over-expression fragments and the Cas9 and sgRNA expression cassette to the wild-type Starmerella bombicola.
After incubation and fermentation, test the yield and acid/lactone ratio of sophorolipids produced by different strains.
After over-expressing UGTB, the yield could be increased a lot than that of the control.
BBa_K3402056: Single-gene editing cassette
This device is composed of 50bp-upPXA1 (BBa_K3402037), hph (BBa_K3402012), 50bp-doPXA1(BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), Tsyn7(BBa_K3402001).
We achieve the knockout of PXA1 gene and insert the hygromycin resistant gene as a selection marker. Two parts of PXA1 are homologous arms at the ends. The strongest promoter Ptef1 is used to express sgRNA to guide Cas9 protein to edit PXA1 site.
The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the PXA1 site.
The single gene-editing efficiency was 100%.
BBa_K3402057: double-gene editing cassette
This device is composed of 50bp-upPXA1 (BBa_K3402037), hph (BBa_K3402012), 50bp-doPXA1 (BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), sgGFP (BBa_K3402024), Tsyn7 (BBa_K3402001), upGFP (BBa_K3402025), doGFP (BBa_K3402026).
We knocked out the PXA1 and GFP gene by this device. In the meantime, we inserted hygromycin resistance gene at the origin PXA1 site by homology directed repair, which was to used as the marker gene to know if it is successful to knock PXA1.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were extracted genomes for PCR, gel electrophoresis analysis and conducted green fluorescence observation. As a result, all of the target fragments were verified to be right in gel electrophoresis analysis and only two positive colonies showed fluorescence.
The double gene-editing efficiency was 99%.
BBa_K3402058: Triple-gene editing cassette
This device is composed of up-doLeu (BBa_K3402048), sgLeu (BBa_K3402049), 50bp-upPXA1 (BBa_K3402037), hph (BBa_K3402012), 50bp-doPXA1 (BBa_K3402038), Ptef1 (BBa_K3402007), sgPXA1 (BBa_K3402039), sgGFP (BBa_K3402024), Tsyn7 (BBa_K3402001), upGFP (BBa_K3402025) and doGFP (BBa_K3402026).
Based on Double-gene editing expression cassette, we designed the sgRNA that targeted at Leu site. We constructed the Pxa1, GFP, Leu editing expression cassette, and transformed it into the recombinant strain.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colonies grown on the CM but did not grow on the MM were the correct transformants.
The triple gene-editing efficiency was 30%.
|2||BBa_K3402051||Device||yeGFP expression cassette with NLS||2300|
|3||BBa_K3402053||Device||Self-excising hygromycin marker cassette||4305|
|4||BBa_K3402054||Device||Over-expression SBLE cassette||7061|
|5||BBa_K3402055||Device||Over-expression UGTB cassette||1479|
|6||BBa_K3402056||Device||Single-gene editing cassette||2663|
|7||BBa_K3402057||Device||Double-gene editing cassette||3448|
|8||BBa_K3402058||Device||Triple-gene editing cassette||4500|