Oct 24, 2020
pYCR cloning strategy
- Andreea S1
- 1University of Groningen
- iGEM Groningen 2020
Protocol Citation: Andreea S 2020. pYCR cloning strategy . protocols.io https://dx.doi.org/10.17504/protocols.io.bnvxme7n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
Protocol has been developed based on literature, but hasn't been tested yet.
Created: October 23, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 43671
Keywords: pycr cloning strategy, crispr vector, synthetic dna for yqcg, using crispr, nlp14a in the genome, pcr product of gamy-fw, crispr in place, crispr, synthetic dna, using t4 ligase, pycr-gamy, switch cloning, backbone of pycr, cytoplasmic plasmid, pycr-gamy vector, gtrpeyqcg plasmid, t4 ligase, antitoxin mechanism, nlp14a sequence, genome, bacillus mycoides by use, pycr-gtrp, bacillus mycoide, resulting pcr product, pycr-gtrpe vector, pycr, introducing nlp14a
Abstract
Introducing NLP14a in the genome of B. mycoides: In order to create plasmid pYCR-gamyNLP, the backbone of Pycr will be digested with the PCR product of gamy_Fw and gamy_Rv to produce the pYCR_gamy (CRISPR vector containing the gRNA). The NLP14a sequence will be ordered from Twist Biosciences And PCR-amplified with the primers NLP_Fw and NLP_Rv. The resulting PCR product, as well as the pYCR_gamy vector will be digested with SfiI. The digested mixtures will be ligated using T4 ligase.
Kill switch cloning: The suggested kill switch mechanism is based on Trp auxotrophic strains as well as a toxin-antitoxin mechanism. The toxin will be introduced in the genome using crispr in place of trpE. The antitoxin will be maintained as a cytoplasmic plasmid (pAD-YqcF) and expressed only in the presence of solanine. Unfortunately, to the best of our knowledge, no operator that binds solanine of B. mycoides has been described in literature. If it is provebn that no such operator is present, we suggest on oly keeping the tryptophan dependence strategy.
In order to create plasmid pYCR-gtrpEYqcG plasmid, the backbone of Pycr will be digested with BsaI and ligated with the annealing product of gtrpe_Fw and gtrpe_Rv to produce the pYCR_gtrp (CRISPR vector containing the gRNA). Synthetic dna for yqcG will be ordered from Twist Biosciences and PCR-amplified with the primers Yqcg_Fw and Ygcg_Rv. The resulting PCR product, as well as the pYCR_gtrpE vector will be digested with SfiI. The digested mixtures will be ligated using T4 ligase
Protocol was adapted after a paper of Yi et al., 2018
1. Yi, Y., Li, Z., Song, C. & Kuipers, O. P. Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environ. Microbiol. 20, 4245–4260 (2018).
Clone sgRNA sequence into pYCR
Design sgRNA spacer sequence (~20 nts) using "Benchling'" and choose B. mycoides M2E_15 genome
Choose the gRNA with the highest on-target score and the lowest off-target score.
Order the 2 complementary oligos flanked by overhang containing BsaI restriction site
Anneal oligos by mixing the following components:
-70 µL Nuclease Free water
-10 µL Oligo DNA annealing buffer 10x (100 millimolar (mM) Tris 8 , 500 millimolar (mM) NaCl, 10 millimolar (mM) EDTA)
-10 µL DNA oligos A and B (10 micromolar (µM) each)
Mix well and incubate for 00:05:00 at 90 °C
5m
Cool slowly to room temperature (aprox. 01:00:00 ) by removing the heat block from the apparatus
1h
Run agarose gel (1%) to check the annealing product
Digest pYCR with BsaI and inactivate the enzyme by PCR clean up
Ligate the annealing product with the precut pYCR:
MIx:
-10 µL l of annealing product
-60 ng of digested pYCR
-1 µL T4 ligase
Transform the ligation mixture into E. coli MC1061 using the heat-shock methos.
Grow the cells on LB agar plates + Erithromycin 0.1 mg/mL
To select the transformants perform a colony PCR with Fw primer binding upstream of PvanP and Rv primer downstream SfiI cloning sites.
Expected result
Succesful transformation will have a size of 427 pb
Miniprep and sequence the constructs
Clone flanking regions into pYCR containing sgRNA
Select the repair fragment of choice:
-for knock-out: PCR amplify aprox. 1 kpb upstream and downstream of the sgRNA targeted region
-for knock-in: sequence of the desired inserction sequence flanked by SfiI restriction sites compatible with the backbone of pYCR
Digest the pYCR(+sgRNA) and the repair fragments with SfiI
Inactivate SfiI by PCR clean up
Ligate the components using T4 ligase
Heat inactivate the ligase (incubate 00:20:00 60 °C )
20m
Perform SmaI digestion to get rid of the original pYCR
Transform the ligation mixture into E. coli MC1061 using the heat-shock methos.
Grow the cells on LB agar plates + erythromycin
To select the transformants perform a colony PCR with Fw primer binding upstream of PvanP and Rv primer downstream SfiI cloning sites.
Miniprep and sequence the constructs
Transform final construct in B. mycoides M2E_15
Transform using an electroporation protocol using 5 µL plasmid DNA
Pick 1 colony of B. mycoides M2E_15 and inoculate it in BHIS
When OD600nm reached 0.85 add 2% glycine and 2% threonine in order to weaken the cell wall.
Grow the cells overnight at 200 rpm, 30°C
Dilute the overnight culture 50 times in LBSP medium until the OD600nm reaches 0.65
Collect the cells by 4000 x g, 4°C, 00:10:00 . Discard supernatant
Wash cell pellet with pre-chilled electroporation buffer (10% glycerol, 0.25 M sorbitol, 0.25M trehalose) x4
Suspended in 1 ml electroporation buffer (10% glycerol, 0.25 M sorbitol, 0.25M trehalose).
Snap-freeze the electrocompetent cells in liquid nitrogen and sore at -80 °C
For the PCR mix pipette:
- 5 µL primers mixture Fw + Rv ( 5 uM) (see table ''Primers'')
- 10 µL HF buffer
- 1 µL dNTP (10mM)
- DNA template (0.1 ng)
- 0.25 µL phusion polymerase
- MIliQ to 50 µL
PCR reaction is performed with the following protocol:
| Temperature (*C) | Time (mm:ss) | |
| 98 | 05:00 | |
| 98 | 10:00 | |
| Variable | 00:30 | |
| 72 | 00:30 | |
| repeat above steps | 30x | |
| 72 | 10:00 | |
| 11 | Infinite |
Load 5 µL of sample plus 1 µL of staining solution 6x. Check the length of the band (bp).
Purify sample : PCR cleanup/ gel extraction according to manufacturer.
Digest the polymerized insert and the desired vector with 2U of apropriate restriction enzyme. See table ''Primers'' in order to choose the enzyme.
Digestion mixture (20 µL ):
-SfiI 2 U
- Plasmid (50 ng )
-Insert (20 ng )
-2 µL CutSmart 10x (or appropriate buffer)
-MiliQ to 20 µL
Incubate 01:00:00 at the temperature recommended by the manufacturer of the restriction enzyme
Inactivate the restriction enzyme by incubationg 20 min 65 °C
**SfiI can't be heat inactivated so in order to inactivate it a PCR clean up is necesary
Ligate the digested insert and vector.
Ligation mixture :
-10 ul digestion product
-1 ul T4
-2 ul T4 ligase buffer
-7 ul MQ
Incubate 02:00:00 Room temperature
Thaw on ice 100 µL of electrocompetent cells
Add 2 µg of plasmid and the aliquot of electrocompetent cells to ice-cold electroporation cuvettes
Electroporate 25 uF, 10 kV/cm, 200 Ohms
Add 1 ml of BHIS and incubate 100 rpm, 30°C, 05:00:00 for recovery
Plate on LB + Agar (1.5%) + 100 μg/ml spectinomycin.
For crispr cloning, to activate cas9 expression, add 0.2% mannose.
Incubate at 30 °C Overnight
Randomly pick potential mutants and test by colony PCR (for primers use the ones that flank the region of insertion sites).
After selecting colonies that show the expected band size, purify the desired plasmid (miniprep) and transform into B. mycoides.
Plate on LB agar containing erythromycin
Grow at 28 °C overnight
Pick colonies and test them by colony PCR
If the expected band size is present, purify the plasmid and sequence
Grow the colonies with the expected sequence on liquid LB + erythromycin + manose
Incubate 200 rpm, 28°C, 12:00:00
Next day pick colonies in BHI medium (without antibiotic) and grow at 37 °C overnight
Dilute the liquid cultures and plate them on LN (no antibiotic)
grow overnight st37 °C