Revision as of 01:27, 30 October 2017

Materials

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)

Plasmids	Purposes	Cite/Source
pSPAsp-hp	Shuttle vector E. coli/B. meg; Protein production	pSPAsp-hp was a gift from Dieter Jahn (Addgene plasmid # 48122)
pBE-2	Shuttle vector E. coli/B. subtilis; Protein production	pBE-2 was obtained from China Center for Type Culture Collection(CCTCC)
pBE-p43	Shuttle vector E. coli/B. subtilis; Protein production	pBE-p43 was obtained from CCTCC
pET28a	Protein production in E. coli	pET28a was obtained from Room 6133 of College of Life Sciences, Wuhan university(CLS-WHU)
pET26b	Protein production in E. coli	pET28a was obtained from Room 6135 of CLS-WHU

Strains	Purposes	Cite/Source
Escherichia coli DH5α	Plasmid replication, Protein production	E. coli DH5α was obtained from Room 6133 of CLS-WHU
Bacillus subtilis 168	Protein production	B. subtilis 168 was obtained from CCTCC
Bacillus megaterium DSM B32	Protein production	B. megaterium DSM B32 was obtained from CCTCC
Nitratireductor pacificus pht-3B	Genome PCR	Nitratireductor pacificus pht-3B was obtained from CCTCC

Primer names	Sequence
Prefix-PartA-F	5’- TCTAGAGGCCGCTGCGATCCCGCGAAG-3’
PartA-RBS-R	5’- CTCCTCTTTAATCTCTAGACATCCTCTATGGTACTCGTGATGGCTTTATTG -3’
RBS-Rdh-F	5’- TCTAGAGATTAAAGAGGAGAAATACTAGATGAGACTTTACAGCAATAGAGATAGACCGAA-3’
Rdh-Ter-R	5’- GTTTTATTTGATGCCTGGTTATTATCCCGCTGATGATTTAAGTCTTG-3’
Rdh-Ter-F	5’- ATCATCAGCGGGATAATAACCAGGCATCAAATAAAACGAAAG-3’
Ter-Postfix-R	5’- CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGC -3’
pSPAsp-hp-BsrG1-F	5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT -3’
pSPAsp-hp-BamH1-R	5’- GCCGGTACCGGATCCTTATTATCCCGCTGATGATTTAA -3’
pHIS1525-BsrG1-F	5’- AAAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’
pHIS1525-BamH1-R	5’- CCGGTACCGGATCCTTATTATCCCGCTGATGATTT-3’
pHIS1525-2-F	5’- AAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’
pHIS1525-2-R	5’- CCGGTACCGGATCCGTCCCGCTGATGATTT-3’
pSPAsp1-F	5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT-3’
pSPAsp1-R	5’- GCCGGTACCGGATCCGTCCCGCTGATGATTTAA-3’

(Not all the primers are listed here but some important ones)

@@ Line 184: / Line 184: @@
 <table border="1" align="center">
 	<tr bgcolor="#bdbbdb"><td style="text-align:center;vertical-align:middle;">Primer names</td>
-	<td style="vertical-align:middle;vertical-align:middle;">Sequence</td></tr>
+	<td style="text-align:center;vertical-align:middle;">Sequence</td></tr>
 	<tr><td style="text-align:center;vertical-align:middle;">Prefix-PartA-F</td>
 	<td style="vertical-align:middle;">5’- TCTAGAGGCCGCTGCGATCCCGCGAAG-3’</td></tr>