Team:WHU-China/Experiments

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Experiment

• 1 Materials
•      1.1 Solutions     1.2 Plasmids
•      1.3 Bacteria     1.4 Primers
• 2 Methods
•      2.1 General construction of xylose-inducible expression vectors
•      2.2 Transformation of B. megaterium with these plasmids
•      2.3 Induction of protein production
•      2.4 Optimization of RBS
•      2.5 Integration of hemAKK and xylA Promoter upstream of Operon hemAXCDBL
•      2.6 Coexpression CBD and MAD
•      2.7 Introduction of engineered B. megaterium into HMBR
•      2.8 Detection and adjustment with Model

Materials

1.Solutions

Solutions	Ingredients	Sterilization
2x AB3(Antibiotic Medium No.3)	35g/L AB3(DIFCO) in dH2O	Autoclave at 121℃ for 20 min
2×SMM	40mM Maleic acid, 80mM NaOH, 40mM MgCl2•6H2O, 1M Sucrose in dH2O	Filtration
PEG-P	400g/L PEG-6000 in 1x SMM	Autoclave at 121℃ for 11 min
SMMP	2x AB3 : 2×SMM= 1:1	Separately sterilized
Solution A	206g/L Sucrose, 13g/L MOPS, 1.2g/L NaOH in dH2O.Adjust to pH 7.3 with NaOH	Filtration
Solution B	14.04g/L Agar, 0.70g/L Casamino acids, 35.09g/L Yeast Extract in dH2O	Autoclave at 121℃ for 15 min
8x CR5 salts	2g/L K2SO4, 80g/L MgCl2•6H2O, 0.4g/L KH2PO4, 17.6g/L CaCl2 in dH2O	Autoclave at 121℃ for 15 min
12% Proline 120g/L	L-proline in dH2O	Filtration
20% Glucose	200g/L Glucose in dH2O	Filtration
CR5-top agar(2.5ml)	1.25ml Solution A, 288μl CR5 salts, 125μl 12% Proline, 125μl 20% Glucose, 713μl Solution B(42℃)	Separately sterilized and mix in order
Lysis buffer	100mM Na3PO4, 5mg/ml Lysozyme. Adjust to pH 6.3 with H3PO4. 1μl 1 M MgSO4 solution and 2 μl HS-Nuclease* (250 U/μl, cat.# GENUC10700-01, final concentration of 500 U/ml)	Filtration

(Other basic molecular biological materials are not listed here)