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results_group12

Group12

Introduction

To realize hair dyeing & perming and decoloring & straightening as designed, we use E.coli to construct all the needed plasmid and transfect them into yeast through electroporation to express target enzymes and peptides. To confirm the validity of our perming and dyeing theory and eventually, the whole project, real hair is used in lab trails, which aim to estimate its color fastness, mechanical property, etc.

Plasmid construction and amplification

The transformation of plasmid with AOX1 as promoter

First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.

Fig1. Colony PCR confirmation of successful E.coli transfection

Bright bands of identical sizes from colony PCR result demonstrates that target plasmid had successfully transformed into E.coli

Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.

Fig2. Colony PCR result of AOX1-α factor-ROX1-AOX1 Terminator transformed E.coli

The band of AOX1-α factor-ROX1-AOX1 Terminator from colony PCR is about 3000bp, identical to the theoretical length of 3070bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully transformed into E.coli

Fig3. Colony PCR result of AOX1-α factor-Laccase-AOX1 Terminator transformed E.coli

The band of AOX1-α factor-Laccase-AOX1 Terminator from colony PCR is about 3000bp, identical to the theoretical length of 3416bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully transformed into E.coli

Fig4. Colony PCR results of AOX1-α factor-FMO-AOX1 Terminator, AOX1-α factor-crtE-AOX1 Terminator, AOX1-α factor-crtB-AOX1 Terminator and AOX1-α factor-crtI-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1 Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

Fig5. Colony PCR results of AOX1-α factor-CUS-AOX1 Terminator, AOX1-α factor-ACC-AOX1 Terminator, AOX1-α factor-4CL-AOX1 Terminator and AOX1-α factor-LOX2-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator (3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from colony PCR are identical to the theoretical lengths of 3046bp, 3619bp, 3523bp and 4528bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

Fig6. Colony PCR results of AOX1-α factor-curA-AOX1 Terminator, AOX1-α factor-pepACS-AOX1 Terminator and AOX1-α factor-DsbC-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-curA-AOX1 Terminator (almost 3000bp), AOX1-α factor-pepACS-AOX1 Terminator (almost 2000bp) and AOX1-α factor-DsbC-AOX1 Terminator (almost 3000bp) from colony PCR are identical to the theoretical lengths of 2875bp, 1987bp and 2722bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

The construction of plasmid with Panb1 or Pynr071c as promoter

AOX1 promoter is the strongest eukaryotic promoter currently known in yeast expression system. So we choose AOX1 as the primary promoter when we synthesized all these plasmid for the sake of more convenient expression. But noticing that methanol is hazardous, flammable, combustible and therefore, inappropriate to have direct contact with the hair, we need to substrate AOX1 for constitutive promoter Panb1 and xylose induced promoter Pynr071C to realize the projected regulation function as designed. Double-enzyme cleavage and rejointing is used to achieve this. We amplify the target gene located in the primarily synthesized plasmid without AOX1 promoter, and digest the acquired fragments and two kinds of plasmid, containing promoter only, with EcoR I and BamH I, then transform them into E.coli after linking the product together. Through this, we successfully substrate the primary AOX1 for Panb1 and Pynr071C.

Fig7. Plasmid construction and colony PCR results of reconstructed plasmid with Panb1 and Pynr071C promoter

All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.

The construction of plasmid without signal peptide

During the experiment, we find that not all the protein we want could be secreted extracellularly. Target protein is only detected in the yeast but not in the culture. while no signal appears in the supernatant without them, which indicates that not all of our signal peptides works. We reckon that as the α-factor on the plasmid we used is only a common basic signal peptide, which has some degree of universality but couldn’t fit all the protein due to unseen matters like space structure of the protein, our target protein couldn’t be induced to secret into the extracellular space by α-factor, or the low expression level coupled with degradation from protease existing in the extracellular environment could do the same. So, to get the rest of our enzymes which don’t want to be outside of the cell with α-factor, and whether or not we could raise the expression level, we design new primers for PCR, which eliminate the signal peptide part off the whole target genes, and reconstruct the plasmid without the α-factor to verify if they prefer to be expressed inside of the cell.

Fig11. Plasmid construction and colony PCR results of plasmid without signal peptides transformed E.coli

All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.

Exploring the construction of plasmid for pigment synthesis

After all the expression product is detected, to explore the molecular mechanism laid behind and to save more experience for future teams, we tried to construct the complete whole pathways. Using seamless cloning, tentative experiments are performed by new primer for homologous recombination.

Unfortunately, no plasmid with multiple target genes are constructed. Good news is double-target-gene plasmid obtained. It seems that the identical promoters and terminators lead to the much shorter product of seamless cloning than theoretical, which means the enzymes used to join the designed homologous arms—by primers— falsely take some of the shared promoters and terminators as substrates as well. Then we give double-enzyme digestion and reconnection a try but limited to the restriction sites existed in our target genes and ‘more fragments less odds’ and, well, time, no complete pathway is constructed. What we do have now is the precious experimental experience for all of our team members, and useful information for future teams.

Electroporation and expression of yeast

To verify the expression of our genes and to acquire corresponding enzymes, we choose Pichia Pastoris GS115 as chassis and using electroporation to blend our plasmids into them. Large amount of target gene containing plasmid is extracted, then we digest them with Bgl II or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Concentration is also applied to achieve higher copy number and higher expression level.

After electroporation of plasmid with AOX1 as promoter and those without signal peptide, we acquire identical bands through Nickel-affinity chromatography column and SDS-PAGE.

For some of our enzymes don’t have standard protocol to estimate their activity at present, we add substrates into culturing medium accordingly to find out whether there exists active target enzymes and do get our indigo and lycopene synthesized.

Fig16. Medium for expression with substrates From left to right:

GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole; mixture of Panb1-crtE-AOX1 Terminator、Panb1-crtB-AOX1 Terminator、Panb1-crtI-AOX1 Terminator medium with FPP

Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Fig17. Colony PCR result of yeast after electroporation

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide. At the same time, because the molecular weight of PepACS is only 5.08 kDa, it is almost impossible to separate from bromophenol blue, and its migration is also greatly affected, so the position of the band is not accurate. In order to judge whether it was expressed or not, we first filtered the miscellaneous proteins through 10kDa ultrafiltration tube, then concentrated it with 3kDa ultrafiltration tube, and then detected it by SDS-PAGE. Judge whether it is expressed successfully by whether there is coloring near the bottom line.

Fig18. SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS detecetion in the supernatant

Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa; DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression. There are irregular staining bands on the bromophenol blue line in the PepACS swimming lane. Although the molecular weight is not accurate, the successful expression can be judged according to the detection method we designed.

After confirmation of successful secret of some of the protein, we test the enzymatic activities of laccase and LOX2, which have standard activity testing protocol. Because of the thin band of laccase from SDS-PAGE, we can’t be entirely sure whether it’s inactive or active but too low the concentration to be detected. So, purification through Nickel-affinity chromatography column is used to raise its concentration for further test of enzymatic activity.

Fig19. SDS-PAGE result of laccase after purification through Nickel-affinity chromatography column

Clear and thick band of about 75kDa is consistent to the result in the supernatant. Higher the eluent concentration, thicker the band. This indicates that the band of laccase in Fig18 is our target protein instead of other impurity with a high expression level.

During the test, we find a vague band of ROX1 which couldn’t be verified as successfully expressed according to its SDS-PAGE result in the supernatant. As the key component of our xylose responding system, the successful expression of ROX1 is most vital to our project, so we apply purification through Nickel-affinity chromatography column to ROX1 to further identify its expression. Lucky us, target band is acquired and confirmed. It feels so good to announce that our ROX1 can be expressed like the others.

Fig20. SDS-PAGE result of ROX1 after purification through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 55-55kDa, bigger than the theoretical 47.09kDa but still within explainable and acceptable range of glycosylation modification. ROX1 could be confirmed as successfully expressed. Although no gradient elution is applied, following elution result could verify this is ROX1, with some impurity bands occurring in the first ROX1 elution.

At the same time, after several SDS-PAGE confirmation, some of our proteins still don’t want to show themselves. No band detected. We infer that this is due to A. the low level of extracellular expression and degradation by protease existing in the extracellular environment and B. the common signal peptide we use—α-factor—isn’t specialized according to the unique space structure of our proteins so the mismatch causes failure of extracellular expression. To solve this, we reconstruct plasmids without the signal peptide and try to do intracellular expression. This is aim at all the undetectable or low-expressed genes.

Fig21. Colony PCR result of yeast after electroporation of reconstructed plasmid without the signal peptide

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.

After verification of successful transfection, we can’t test the protein directly due to intracellular expression. So, we extract the total protein in yeast and go for a purification through Nickel-affinity chromatography column, then apply SDS-PAGE to separate target protein from the large amount and various type of total protein to confirm whether our target protein could be expressed and value its expression level quantitatively.

Fig22. SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO,

Fig23. SDS-PAGE result of crtE after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 33.42kDa but still within explainable and acceptable range of glycosylation modification. crtE could be confirmed as successfully expressed.

Fig24. SDS-PAGE result of crtB after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 35.30kDa but still within explainable and acceptable range of glycosylation modification. crtB could be confirmed as successfully expressed.

Fig25. SDS-PAGE result of crtI after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 55.86kDa but still within explainable and acceptable range of glycosylation modification. crtI could be confirmed as successfully expressed.

After confirmation of successful expression, we add indole as substrate into culture medium of FMO and FPP into the mixture of crtE, crtB and crtI to test whether the expressed enzyme is active in terms of color change of the culture medium. As for the result, we succeed in expression of enzymes and synthesis of indigo and lycopene.

Fig26. Medium for expression with substrates

From left to right:

GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole; mixture of Panb1-crtE-AOX1 Terminator、Panb1-crtB-AOX1 Terminator、Panb1-crtI-AOX1 Terminator medium with FPP

Determination of enzyme avtivity

After the target strips appeared on SDS-PAGE, for the enzymes with standard enzyme activity assay such as Laccase and LOX2, we measured their enzyme activity according to the standard measurement methods to detect whether our enzymes are catalytically active. Laccase and LOX2 show high activity after adjusting the relevant conditions such as pH, temperature and ion concentration

Verification of promoters

To verify whether our promoter could initiate the expression of downstream genes, we linked GFP to the Panb1 and Pynr071C promoter, then transfect them into yeast and test the exist of fluorescence.

Team:HUST-China/results group12