Experiments
Plasmid construction:
The first stage: construct plasmid pOdd-1, pOdd-2, pOdd-3 at the same time
The second stage: pOdd-1 and XynA connection to obtain pXlnA, pOdd-2 and XynB connection to obtain pXlnB, pOdd-3 and XynD connection to obtain pXlnD
The third stage: construct pXylan-B, pXylanBD, pXylaN (*N=BDA)
The fourth stage: transformation of yeast. The obtained pXylan-B, pXylanBD, and pXylaN were extracted from E. coli and transformed into yeast for the next functional test.
Function test: Fermentation performance test-test sugar and alcohol production capacity
PCR experiment:
PCR experiment can be divided into three steps: denaturation → annealing → extension. Denaturation: heating breaks the hydrogen bond between double strands of template DNA at a high temperature of 95 ℃ to form two single strands. Annealing: reduce the solution temperature by 60 ℃, and make the template DNA and primer complement each other according to the principle of base pairing. Extension: when the reaction temperature of the solution rises to 72 ℃, the heat-resistant DNA polymerase takes the single strand DNA as the template and replicates the complementary DNA in the direction of 5 '- 3' by using four kinds of deoxynucleoside triphosphate (dNTP) in the reaction mixture under the guidance of primers. We also did pre denaturation and complete extension to completely denature and untie the primers, so as to ensure that the primers are single stranded DNA, so as to ensure the effect. The final extension is to fully extend the primers and completely anneal the single stranded products to form a double stranded structure to ensure the effect.
Gel electrophoresis experiment:
TAE buffer was added and agarose gel was added to the template, waiting to be condensed into solid, then put into TAE buffer and added DNA reagent of mixed nucleic acid dye into the gel electrophoresis apparatus to wait for the result. The results can be seen visually under UV lamp.
LB medium:
The medium is used to pre culture the strains to multiply the strains and meet the use requirements. It can also be used to culture genetically engineered receptor bacteria. We cultured E. coli.
Plasmid transformation by heat shock method:
Plasmid transformation refers to the process of introducing our plasmid into bacteria. The connected products were put into competent cells. It is convenient for subsequent molecular operation. To achieve the proliferation of recombinant clones.
Hygromycin plate screening:
The plate contains hygromycin, and our plasmid has the function of anti hygromycin. Only the yeast transferred into our plasmid can grow on the plate. At the same time, many methods can be used to screen and identify the target clones.
pXlnB plasmid construction:
pXlnB is GAP promoter+AnXlnB orf+CYC1 terminator transcription unit plasmid.
GAP promoter part acquisition: Using Saccharomyces cerevisiae DNA (or colony) as a template and primer pair GAPp-F1/GAPp-R1 to amplify the GAP promoter part, with a size of 695bp.
AnXlnB orf part acquisition: Using the synthesized AnXlnB orf sequence as a template and the primer pair AnXlnBorf-F1/AnXlnBorf-R1 to amplify to obtain AnXlnB orf part with a size of 706bp.
Obtained CYC1 terminator part: Using Saccharomyces cerevisiae DNA (or colony) as template and primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part. The size is 276bp.
pOdd-1 plasmid acquisition: Using pAR318 plasmid as template and primer pair pOdd-1-F1/pOdd-1-R1 to amplify to obtain 1757bp fragment. Then use this fragment as template and pOdd-1-F2/pOdd-1-R2 primer to amplify to obtain a 1804bp fragment. This 1804bp fragment has repetitive sequences at both ends. The linear fragment can be ligated into a circular plasmid using Gibson or a recombination kit (Novizan), and screened on a kanamycin plate.
The obtained pOdd-1 plasmid, GAP promoter part, AnXlnB orf part, CYC1 terminator part were cloned by Bsa1 restriction enzyme digestion and ligation, and kanamycin plate screening was performed. The final plasmid was named pXlnB.
pXlnD plasmid construction:
pXlnD is a TPI1 promoter+AnXlnD orf+CYC1 terminator transcription unit plasmid.
TPI1 promoter part acquisition: Use Saccharomyces cerevisiae DNA (or colony) as a template and primer pair TPI1p-F1/TPI1p-R1 to amplify the TPI1 promoter part, which is 614bp in size.
AnXlnD orf part acquisition: Use the synthesized AnXlnD orf sequence as a template and the primer pair AnXlnDorf-F1/AnXlnDorf-R1 to amplifyto obtain AnXlnD orf part with a size of 2443bp.Obtained CYC1 terminator part: Using Saccharomyces cerevisiae DNA (or colony) as template and primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part. The size is 276bp.
pOdd-2 is the TPI1 promoter+AnXlnD orf+CYC1 terminator transcription unit receiving plasmid: the pAR318 plasmid is used as the template, and the primer pair pOdd-2-F1/pOdd-2-R1 is amplified to obtain a 1757bp fragment. Use this fragment as a template, pOdd -2-F2/pOdd-2-R2 primer to amplify1804bp fragment. This 1804bp fragment has repetitive sequences at both ends. The linear fragment can be ligated into a circular plasmid using Gibson or a recombination kit (Novizan), and screened on a kanamycin plate.
The obtained pOdd-2 plasmid, TPI1 promoter part, AnXlnD orf part, CYC1 terminator part were cloned by Bsa1 restriction enzyme digestion and ligation, and kanamycin plate screening was performed. The final plasmid was named pXlnD.
pXynA plasmid construction:
pXynA is FBA1 promoter+AnXlnD orf+CYC1 terminator transcription unit plasmid.
Obtained FBA1 promoter part: Use Saccharomyces cerevisiae DNA (or colony) as template and primer pair FBA1p-F1/FBA1p-R1 to amplify to obtain FBA1 promoter part, with a size of 848bp.
Obtained CcXynA orf part: Use the synthesized CcXynA orf sequence as a template and the primer pair CcXynA orf-F1/ CcXynA orf-R1 to amplify to obtain the CcXynA orf part with a size of 1591bp.
Obtained CYC1 terminator part: Use Saccharomyces cerevisiae DNA (or colony) as template and primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part, the size is 276bp.
pOdd-3 plasmid acquisition: Use pAR318 plasmid as template and primer pair pOdd-3-F1/pOdd-3-R1 to amplify to obtain 1757bp fragment. Then use this fragment as template and pOdd-3-F2/pOdd-3-R2 primer to amplify to obtain a 1804bp fragment. This 1804bp fragment has repetitive sequences at both ends. The linear fragment can be ligated into a circular plasmid using Gibson or a recombination kit (Novizan), and screened on a kanamycin plate.
The obtained pOdd-3 plasmid, FBA1 promoter part, CcXynA orf part, CYC1 terminator part were subjected to one-step cloning with Bsa1 restriction enzyme digestion, and kanamycin plate screening, and the final plasmid was named pXynA.
Transformation
The ligation product is transformed into DH5a competent cells and screened on LB/kana plate
Colony PCR System
Identification primer cx-podd-kana-F/ CYC1t-R1, 2*mix, 55 degrees annealing, 72 degrees extension for 4 minutes
Send to company for sequencing
pXylan-B plasmid construction:
Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid) and the primer pair Backbone-XlnB-F1/Backbone-R1 to amplify the 5479bp Backbone-XlnB fragment, which is recovered from the gel and digested with Sap1 for use.
The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use Sap1 restriction enzyme digestion.
The above two fragments, namely the Backbone-XlnB fragment after Sap1 digestion, and the 1624bp fragment, were ligated and cloned by restriction enzyme digestion, and screened on ampicillin plates. Finally, the plasmid pXylan-B is obtained.
pXylan-BD plasmid construction:
Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid) and the primer pair Backbone-XlnBD-F1/Backbone-R1 to amplify the 5479bp Backbone-XlnB fragment, which is recovered by gel and digested with Sap1 for use.
The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use Sap1 restriction enzyme digestion.
The pXlnD plasmid was digested with Sap1 to obtain a 3280+1727bp fragment, and the 3280bp band was recovered for use.
The above three fragments, namely the Backbone-XlnBD fragment after Sap1 digestion, the 1624bp fragment, and the 3280bp fragment, were cloned by restriction digestion and ligated in one step, and screened on ampicillin plates. Finally, the plasmid pXylan-BD was obtained.
pXylan plasmid construction:
Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid). The primer pair Backbone-F1/Backbone-R1 was used to amplify the 5479bp Backbone fragment, which was recovered from the gel, and digested with Sap1 for use.
The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use Sap1 restriction enzyme digestion.
The pXlnD plasmid was digested with Sap1 to obtain a 3280+1727bp fragment, and the 3280bp band was recovered for use.
The pXynA plasmid was digested with Sap1 to obtain a 2662+1727bp fragment, and the 2662bp band was recovered for use.
The above 4 fragments, namely the Backbone fragment after Sap1 digestion, the 1624bp fragment, the 3280bp fragment, and the 2662bp fragment, were cloned by restriction enzyme digestion and ligated in one step, and screened on an ampicillin plate. Finally, the plasmid pXylan was obtained.
DNS method for the determination of reducing sugars
Steps: Draw 1 mL of sample solution. Put it in a 25 mL colorimetric tube. Add 2 mL of DNS reaction solution, and develop color in boiling water for 2 minutes. Rinse with running water, after cooling quickly, add 9 mL of distilled water to dilute, and shake well. Adjust to zero with distilled water blank, measure the absorbance at 540 nm, and calculate the amount of reducing sugar by referring to the standard curve.
The preparation of the standard curve: the standard glucose is first dried to a constant weight, accurately weighed 1 g, dissolved in a small amount of distilled water, and dilute to 500 mL, so that the reducing sugar concentration is 2 mg/mL. Pipette 0.2, 0.4, 0.5, 0.6, 0.8 mL of standard solution into 25 mL colorimetric tube. Make up to 1 mL with distilled water. Add 2 mL of DNS reaction solution, and develop color with boiling water for 2 minutes. Rinse with running water, and quickly cool down. Dilute with 9 mL of distilled water and shake well. Set the blank with distilled water to zero, and measure the absorbance at 540 nm. Draw the standard curve with the absorbance value OD540 at 540 nm as the abscissa and the reducing sugar content C (g/L) as the ordinate.
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