リム傑威
Lim Jackwee
MultiBac
This is an overview of the MultiBac multigene-polyprotein platform using insect cells to recombinantly obtain large macroassemblies.
From plasmid to protein ~4-5 weeks
Figure. Overview of the MultiBac system for multigene construction. Micrograph showing 72h after cells arrest when infected with bacmid. The YFP count of the infected cells are monitored up to 96h. SDS-PAGE showing polyprotein obtained from 5L of Sf21 cells maintained at 1.6 million cell density recovered at the 72h period. Both YFP and (CFP if included in the polygene construct) counts are monitored.
Reagents needed for Blue-White Screening
Cells
DH10EMBacY E. coli cells (with Kan and Tet resistance and YFP gene for expression)
Sf21 insect cells
Antibiotics (stock at 1000X)
Kanamycin: 50mg/ml
Tetracyclin: 10mg/ml (in absolute ethanol)
Gentamycin: 10mg/ml
Ampicillin: 100mg/ml
Chloroamphenicol: 30mg/ml (in 70% or absolute ethanol)
Other Reagents
IPTG: 1M (1000X)
BluOGal: 100mg/ml (500X) Light sensitive!!!
Sterile LB
Sterile LB agar plates with Kan.Tet.Gent.IPTG.BluOGal
Qiagen MiniPrep Kit
Serum Free Medium for insect cell Sf21 culture
Yellow Fluorescent Protein (YFP) standard
Reagents needed for Cre-LoxP fusion of acceptor and donors
Cre recombinase (from NEB)
10x Cre recombinase buffer (from NEB or home-made)
Standard Ecoli. competent cells (pir- strain: Top10/ OmniMax)
Antibiotics
LB medium
1. For a 10ul Cre reaction, mix 0.5-1.0ul of each educt in equal molar concentrations (or slight excess of donor)
2. Add 1ul 10x Cre buffer and 0.5ul Cre recombinase and adjust to 10ul with MilliQ water
3. Incubate the Cre reaction at 30 (37C) for 1 hour
4. For chemical transformation, mix 5ul Cre reaction with 50ul chemical competent cells. Incubate the mixture on ice for 15-30min and perform heat shock at 42C for 45-60s
For electroporation, 1ul Cre reaction is directly mixed with 50ul electrocompetent cells. The cell/DNA mixture is electroporated using the electroporator at 1.8-2.0kV.
Softwares needed
1. CreRexEMBL-0.2_win32 (in-house program)
2. ApE (from http://biologylabs.utah.edu/jorgensen/wayned/ape/)
Disclaimer
The following material assumes that you are already an experienced MultiBac user and only serves to achieve correctness.
Kan and Tet resistances are provided by the baculovirus genome. A third resistance is introduced by the plasmid you will transform the DH10EMBacY cells with. After you have successfully subcloned your gene of interest into the acceptor and donor plasmids and ready for Cre-LoxP recombination, you will need to setup restriction enzyme screening to identify the correct clones e.g. 1 donor + 1 acceptor obtained from Cre-LoxP fusion setup (above).
Figure. The predicted restriction enzyme digest pattern of TAF5-TAF6 is derived from in-house program CreRexEMBL-0.2_win32 and the public accessible ApE. In the DNA electrophoresis gel, presence of digested fragment size ~5300 bp in TAF5 corresponds to more than one copy and thus discarded.
Day 1: After identifying the correct Cre-LoxP constructs, 1ug of plasmid is transformed into DH10EMBacY on ice (20min), heat-shock 42C (45s), on ice (2min) and 400ul of LB (w/o antibiotic) added for O/N (16h) shaking at 37C.
Day 2: The transformed cells are plated onto sterile LB agar plates with Kan.Tet.Gent.IPTG.BluOGal and left O/N in 37C incubator
Day 3: Both types of colonies may appear; blue and white. Only the white colonies carry the correct plasmid due to loss of galactosidase activity (lacZ gene), following the Tn7 transposition of the gene of interest into the baculoviral genome. Choose the white colonies and restreak for confirmation
Day 4: Confirm that the white colonies are 100% white (very important!!!!). Restreak blue-white clonony screening if needed. Perform MiniPrep and proceed with MultiBac procedures.
For Cloning and Propagation of Bacmids, refer to pir+ strains e.g. DH5alpha and BW23474 cells
For Enzyme Digestion, refer to NEBCutSmart
References:
1. Nature Biotech. 2004 22 (12):1583
2. Curr. Protoc. Protein Sci. 2008 51:5.20.1
3. Genetic code expansion for multiprotein complex engineering. Nat Methods. 2016 Oct 17. doi: 10.1038/nmeth.4032