The λ phage PL promoter is a potent promoter controlled by a temperature-sensitive repressor (cIts857), which can repress transcription of the PL promoter at low temperatures but not at high temperatures. Therefore, the vector with λ phage promoter must be hosted by a strain with cIts857 gene. This experiment is from the next volume of Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook D.W. Russell.
Operation method
Experiments on expression of cloned genes in E. coli using λ phage PL promoter
Materials and Instruments
Vectors and Bacterial Strains PL Expression Vectors Positive Control Plasmids Move makings For more product details, please visit Aladdin Scientific website.
L-tryptophan SDS gel spiking buffer SDS-polyacrylamide gel Target gene or cDNA fragments LB agar plates LB medium M9 basic medium
SorvallGSA turn-table or equivalent Boiling water bath Oscillating incubator
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Caulmers Brilliant Blue Staining Solution or Silver Staining Solution
See Appendix 8.
1XSDS Gel Spiking Buffer
Store 1xSDS gel spiking buffer without DTT at room temperature. 1mol/LDTT storage solution is added to the above buffer as it is used.
L-Tryptophan (10 mg/ml)
Gel
SDS-polyacrylamide gel (10%)
Refer to Appendix 8 for preparation of SDS-polyacrylamide gels for protein isolation.
Nucleic acids and oligonucleotides
Target gene or cDNA fragment
Medium
LB agar plates
LB medium
Different antibiotics are added to the medium depending on the carrier used.
LB medium heated to 65°C
Optional, see step 13.
M9 Basic Medium
Sterilized and supplemented with 0.5% (m/V) glucose, 0.2% (m/V) caseinate hydrolysate and desired antibiotics for tryptophan-induced expression vectors.
Centrifuge and rotor head
SorvallGSA rotor head or equivalent
Special equipment
Boiling water bath
Shaking incubator set to 30°C and 40°C
Required only when using the temperature-sensitive allele of the λ phage cI gene.
Supplemental Reagents
Step 1 of this protocol requires the reagents listed in Chapter 8, Protocol 7.
Step 2 of this protocol requires the reagents listed in Chapter 1, Option 17 or 19.
Step 3 of this protocol requires the reagents listed in Chapter 1, Options 23-26.
Step 4 of this protocol requires the reagents listed in Chapter 12, Option 3.
Vectors and Bacterial Strains
E. coli strains with the cIts857 allele or wild-type allele of the λ phage cI gene are heat-inducible clts857 alleles in M5219 beads and tryptophan-inducible wild-type cI gene in GI724 beads (ATCC55151cI gene).
PL expression vectors (e.g. pHUB,pPLc,pKC30,pASl,pCQV2,pAL-781 and pTrxFus)
Positive control plasmids (PL expression vectors expressing fusion proteins of known size)
Methods
Construction of E. coli strains containing recombinant expression vectors
1. PCR modification (Scheme 7 in Chapter 8) or restriction endonuclease digestion of isolated DNA fragments with restriction enzyme sites at the 5' and 3' ends corresponding to the PL expression vector.
If necessary, a strong ribosome binding site can be placed upstream of the ATG of the cDNA/gene.
To make sure that the amplification reaction does not introduce false mutations, the PCR product should be analyzed for sequence.
2. A DNA fragment of the target cDNA/gene is ligated into the expression vector (Scheme 17 or 19 in Chapter 1).
3. The ligated product is transformed into an E. coli strain containing the cIts857 allele or the wild-type cI gene. The transformants are spread on LB plates containing the appropriate antibiotic of choice (usually 50ug/ml ampicillin) and incubated overnight at 30°C (containing the cIts857 allele) or 37°C (containing the tryptophan-induced wild-type cI gene).
Empty expression plasmids were transformed with the same E. coli strain as a negative control.
4. Screen transformants with insert fragments by colony hybridization and/or restriction enzyme digestion analysis of small quantities of prepared plasmids, oligonucleotide hybridization, or sequence analysis (see Scheme 3 in Chapter 12).
Optimization of inducible target protein expression
Many studies have shown that the rate of cell growth severely affects the expression of exogenous proteins, so it is important to control the amount of inoculated bacteria, the duration of cell growth before induction, and the density of cells after induction. Excessive or excessive growth rates can overburden the bacterial synthesis system and lead to the formation of inclusion bodies.
5. The cI repressor protein can be down-regulated by raising the temperature or adding tryptophan to induce the expression of target proteins, so as to screen the optimal expression conditions.
For the factors affecting the induction of expression, please refer to the note in Step 8 of Scheme 1.
For temperature-inducible systems
a. Pick 1~2 colonies of control and recombinant bacteria respectively, insert them into 1 ml of LB culture medium containing the required antibiotics, and incubate at 30°C overnight. b. For the temperature inducible system, please refer to Step 8 of Scheme 1 for details on the effect of temperature on the expression.
An expression vector encoding a hexosized protein was used as a positive control.
b. Add 50 ul to 10 ml of LB culture medium containing antibiotics and incubate at 30°C in a 50 ml shake flask until mid-logarithmic (A550=0.5?1.0).
c. Pipette lml of uninduced culture into a microcentrifuge tube and process as described in steps 6 and 7 below.
d. Adjust the temperature to 40°C to induce the remaining culture. Continue with step 6.
Although 42-45°C is used to inactivate the λ-phage cIts857 repressor in other sections of this book, 40°C is used here to minimize induction of thermokinetic proteins and to allow for continued bacterial growth.
For the tryptophan-inducible system
a. Pick 1~2 colonies of E. coli containing empty vector and recombinant expression vector (with cI wild-type allele) respectively, insert into 1ml of Supplementary M9 culture medium and incubate at 37°C overnight. b. For the tryptophan induction system, use 42~45°C to inactivate the λ-phage cIts857 repressor.
The expression vector encoding a protein of known size was used as a positive control.
b. Take 50ul into 10 ml of Supplementary M9 culture medium and incubate at 37°C in a 50 ml shake flask until mid-logarithmic ( A550=0.5~1.0).
c. Pipette 1 ml of uninduced culture into a microcentrifuge tube and process as described in steps 6 and 7 below.
d. Add tryptophan to the remaining culture to a final concentration of 100ug/ml and continue incubation at 37°C.
6. After induction for different times (e.g. 1, 2, 4 and 6 h), place 1 ml of sample in a microcentrifuge tube, determine the A550 and centrifuge at high speed for 1 min at room temperature.
7. Suspend the precipitate in 100ul of 1XSDS gel spiking buffer, heat at 100°C for 3 min, centrifuge at high speed for 1 min at room temperature and leave on ice until all samples are processed.
8. Warm the sample to room temperature and apply 40ug or a suspension equivalent to 0.15 OD550 culture to a 10% SDS polyacrylamide gel.
9.8~15V/cm electrophoresis until bromophenolan migrates to the bottom of the separator gel.
10. Observe bands of expression products by staining with komassie blue or silver, or by immunoblotting (see Appendix 8).
There should be no difference in protein banding between untransformed E. coli cells and cells transformed with the empty vector. Recombinant bacteria that have been induced for a certain amount of time should have a band of the expected size.
Bulk expression of the target protein
11. Pick a recombinant E. coli colony and add it to 50 ml of antibiotic-containing LB culture medium or supplemented M9 culture medium.
Incubate overnight at 30°C or 37°C in a 250 ml shake flask.
12. Take 50 ml of the overnight culture and add it to 450 ml of antibiotic-containing LB culture medium or supplemented M9 culture medium and incubate it in a 2L shake flask at 30°C or 37°C with shaking until mid-logarithmic stage ( A550=0.5~1.0), and carry out induction according to step 13 or 14.
13. Induct E. coli with the cIts857 allele by adding 500 ml of medium 13 heated to 65°C. Incubate at 40°C for the optimal time determined by the pre-test. Alternatively, collect the cells by centrifugation at 12000 g (8600 r/min Sorvall's head) for 5 min at room temperature and resuspend the precipitate in 500 ml of LB medium at 40°C. Continue incubation at 40°C for an optimal time as determined by pre-testing.
14. Add tryptophan to a final concentration of 100ug/ml to induce E. coli with the cI allele. continue incubation at 37°C for an optimal time as determined by the pre-test.
15. After the appropriate time of induction, collect the cells by centrifugation at 5000 g (5500r/min Sorvall GSA head) for 15 min at 4°C and continue with the purification protocol:
- If a GST fusion protein is expressed, continue with protocol 5.
- If a GST fusion protein is expressed, proceed to protocol 5. If a maltose-binding protein fusion protein is expressed, proceed to protocol 6.
- If the expression product has a histidine tag, continue to Scheme 7