Compartmentalized self-replication (CSR) is a novel approach to the directed evolution of proteases, especially polymerases. In the simplest form of CSR, it consists of a simple feedback loop in which the polymerase replicates only the gene encoding itself (self-replication). Self-replication occurs in a discrete, separated, non-interacting space formed by a thermally stable water-in-oil emulsion. This experiment is derived from "A Laboratory Guide to Modern Protein Engineering" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Segregated self-replication (a new approach to the directed evolution of polymerases and other enzymes)
Materials and Instruments
E.coli inhibitor strain TG1 Move The following experimental procedure describes in detail the screening of Taq polymerase mutants in E.coli using CSR. This method ensures that the polymerase mutant is active under standard PCR reaction conditions. The methods used to generate libraries are nucleotide analogs [27] and error-prone PCR [28], which are not described here. Primer 1 ( 5'-C A G G A A A A C A G C T A T G A C A A A A A A T C T A G A T A T A AC G AG G G G C AA-3' ) is specific for the expression vector pASK75 [29], and the Taq gene was ligated into the vector pASK75 using XbaI and Sal I. Primer 2 (5'- G TA A A A CGA CGG CCA GTA CC AC CG AAC TG CG GG TG ACG CC AAGC-3' ) is specific for the expression vector PASK75. Each primer contains a 5' protruding sticky end, which allows for enhanced screening of CSRs with primers 3 ( 5'-C AG G AAAC AG C TATG AC-3' ) and 4 ( 5'-GTA A A A CGA CGG CCA GT-3' ). For more product details, please visit Aladdin Scientific website.
Dehydrated tetracycline Tetramethylammonium chloride CSR Oil phase
TYE Flat plate Magnetic stirrer
3.1 Expression of Taq Polymerase in Bacteria with the Tet Promoter
( 1 ) Pick single clones into 2 ml of 2X TY [ see 14.2 ( 2 ) ] medium with 0.1 mg/ml Amp and incubate at 37°C overnight.
( 2 ) Add the above culture solution to fresh 2X TYA at a ratio of 1: 100 and incubate at 37°C until the OD600 of the bacterial solution reaches 0.6~0.9 (the incubation should not be longer than 2~3 h).
( 3 ) Add ACROS (non-toxic tetracycline derivative) at a final concentration of 0.2 μg/ml into the culture medium of step (2) to induce the Tet promoter of pASK75 species and express the protein.
( 4 ) Shake the bacteria at 37°C for 2~4 h.
3.2 Collection of polymerase-expressing cells
( 1 ) Centrifuge the cells in a centrifuge [ 1300 rcf ( 3000 r/min), 15 min ], remove the supernatant, and gently suspend the cells with 1 X Tag buffer in the same volume as the culture medium.
( 2 ) Repeat the centrifugation to collect the cells and resuspend the cells, this time resuspending the cells using 1/10 the volume of culture medium in 1X Taq buffer.
( 3 ) Determine the number of cells by measuring the OD600 absorption value ( OD600 = 1 equals 8 X 108 cells/ml).
3.3 Establishment of CSR
( 1 ) Add the aqueous phase of the CSR to 1X Tag buffer on ice with 1 μmol/L primers 1 and 2, 0.25 mmol/L dNTP, 50 μmol/L TMAC ( optional; see Note 1 ), 0.05% ( V/V ) tryptic ribonuclease free of deoxyribonuclease (optional) and induced expression of the cells (final concentration of 1X109 cells/ml). ml).
( 2 ) Prepare the oil phase CSR by mixing light mineral oil, 4.5% (V/V) Span 80, 0.4% ( V/V) Tween-80, 0.05% ( V/V) Triton X-100 with continuous stirring at room temperature. Due to the high viscosity of the surfactant, it is necessary to prepare a large volume of CSR of the oil phase (greater than 50 ml) and remove the surfactant by cutting off the pipette tip. Once prepared, the oil phase CSR can be stored in the dark at room temperature for up to 1 month.
( 3 ) Add 200 μl of aqueous-phase CSR to 400 μl of oil-phase CSR in a dropwise manner (5~10 μl per drop at 5-s intervals) in a 2-ml flat-bottomed tube, with constant stirring using a magnetic stirrer (1000 r/min).
( 4 ) After the last drop is added, stir continuously for 5 min. the final emulsion should be white and viscous (see Note 2).
3.4 CSR
( 1 ) Transfer the emulsion to a 0.5 ml thin-walled PCR tube (100 μl/tube) and add 2 drops of mineral oil to prevent volatilization (see Note 3).
( 2 ) Carry out the PCR reaction at 94°C for 5 min, lysing the cells and destroying the activity of other enzymes, and then perform 20 cycles as follows: 94°C ( 1 min), 60°C ( 1 min), 72°C ( 5 min).
Upon completion of PCR, the emulsified phase should still be white and covered with an oil phase. There may be a decrease in volume, but this does not indicate that the emulsified compartments are merging. Polymerization or fragmentation of the emulsified phase is directly evidenced by a visible aqueous phase layer underneath the white emulsified phase. This is an indication that a number of factors are interfering with the stabilization of the emulsified phase, such as insufficient mixing of the components.
3.5 Implementation
( 1 ) Separate the aqueous phase of the CSR with 2 times the volume (200 μl) of diethyl ether. vortex the mixture for 20 s and then centrifuge it at 20,000 rcf ( 13,000 r/min) for 2 min.
( 2 ) Two liquid layers appear. Carefully move the aqueous layer below the organic phase and transfer to a new 1.5 ml EP tube.
The CSR reaction can be carried out in two different ways.
3.5.1 PEG extraction method
( 1 ) Extraction of the liquid phase layer with benzoic acid-chloroform (1/1, V/V) followed by chloroform-isoamyl alcohol (24/1, V/V).
( 2 ) To the liquid phase layer, add 0.5 v/v of PEG800/MgCl2 solution [ see 14.2 ( 13 ) ] and mix several times up and down with a pipette.
( 3 ) Centrifuge at room temperature, 20,000 rcf (13,000 r/min) for 10 min. remove the supernatant (containing the remaining primer and dNTP) and resuspend the precipitate in TE [see 14.2(14)].
( 4 ) The amount of residual polymerase may be reduced by adding 20-50 U DpnI to the liquid phase layer and allowing to stand for 1 h at 37°C (see Note 4).
( 5 ) To ensure complete removal of the primers, the CSR product in the liquid phase layer is further purified using the PCR Purification Kit (Qiagen). The product was washed twice with 35% (V/V) guanidine hydrochloride to ensure complete removal of residual primers. Finally, the purified product is added to 50 μl of buffer EB [Qiagen; see 14.2 (16)].
3.5.2 Rapid implementation
( 1 ) Purify the CSR product from the PCR purification kit as described in 14.3.5.1 (step 5). The product is washed twice with 35% (V/V) guanidine hydrochloride to ensure complete removal of residual primers. The purified product was finally added to 50 μl of buffer EB.
(2) Take 7 μl of the product and add 1 μl of 10 X Taq buffer, 1 μl of 20 U/μl DpnI, and 1 μl of ExoSAP, and incubate at 37°C for 1 h, then at 85°C for 15 min.
3.6 Extraction
( 1 ) The gene to be screened is amplified twice using outer nested primers 3 and 4. 20% of the product to be screened is added to 50 μl of PCR reaction solution (i.e., 10 μl of purified product to 50 μl of solution). However, this amount may be increased or decreased depending on the results of the first amplification (see Note). The reaction conditions for the 2nd amplification need to be adjusted according to the results of the first amplification, and 25 cycles are commonly used: 94°C (30 s), 50°C (30 s), 72°C (5 min). The number of cycles can also be adjusted according to the results (see Note).
( 2 ) The results are assayed on an agarose gel. Successful screening is the appearance of a band of the correct size (2.5 kb for the polymerase), whereas no band should appear in the negative control [ see Note 5 and Note 6 ].
The CSR bands can be further purified by excision, re-excised, and recloned into the vector pASK75. It is then transformed into TG1 [ see 14.2 ( 1 ) ], and the transformed product is either screened as described in 14.3.7 or cultured and induced as described in 14.3.2 to 14.3.6 in preparation for the next round of selection.
3.7 Rapid Screening of Taq Polymerase
( 1 ) To screen for mutants, transformed clones are amplified on TYE-Amp plates [ see 14.2 (3)] and then inoculated into 96-well plates containing 100 μl of 2X TYA per well [ see 14.2 (2)].
( 2 ) After 6~10 h of incubation, add 20 μl of 2X TYA containing 1.2 μg/ml ACROS, and continue to incubate for 2~4 h for protein expression.
( 3 ) Using a volley gun, transfer 2 μl of induced cells from each well of the 96-well plate to 30 μl of PCR mixture (see 14.3.3 and 14.3.4), which is also on a 96-well PCR plate.
( 4 ) In a 96-well PCR plate, cover oil is added to each well and the PCR reaction is performed [ see 14.3. 4 (2) ], which can be increased to 30 cycles, and is used to screen for mutants with weaker activity. It is important to use the wild-type as a control. Positive identification can be performed on an agarose gel.