TITLE: pYCR cloning strategy 
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Introducing NLP14a in the genome of B. mycoides: </span><span> 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. </span></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Kill switch cloning: </span><span>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.  </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>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</span><span style = "font-style:italic;"> yqcG</span><span> 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</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Protocol was adapted after a paper of Yi et  al., 2018</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">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). </div></div></div>

[STEPS]
?. [Clone sgRNA sequence into pYCR]
Design sgRNA spacer sequence (~20 nts) using "Benchling'" and choose B. mycoides M2E_15 genomeChoose the gRNA with the highest on-target score and the lowest off-target score.
?. [Clone sgRNA sequence into pYCR]
Order the 2 complementary oligos flanked by overhang containing BsaI restriction site
?. [Clone sgRNA sequence into pYCR]
Anneal oligos by mixing the following components:-  Nuclease Free water- Oligo DNA annealing buffer 10x ( Tris ,  NaCl,  EDTA)- DNA oligos A and B ( each)
70 µl
10 µl
10 µl
?. [Clone sgRNA sequence into pYCR]
Mix well and incubate for  at
90 °C
?. [Clone sgRNA sequence into pYCR]
Cool slowly to room temperature (aprox. ) by removing the heat block from the apparatus
?. [Clone sgRNA sequence into pYCR]
Run agarose gel (1%) to check the annealing product
?. [Clone sgRNA sequence into pYCR]
Digest pYCR with BsaI and inactivate the enzyme by PCR clean up
?. [Clone sgRNA sequence into pYCR]
Ligate the annealing product with the precut pYCR:MIx:-l of annealing product-of digested pYCR-T4 ligase
10 µl
60 ng
1 µl
?. [Clone sgRNA sequence into pYCR]
Transform the ligation mixture into E. coli MC1061 using the heat-shock methos. Grow the cells on LB agar plates + Erithromycin
?. [Clone sgRNA sequence into pYCR]
To select the transformants perform a colony PCR with Fw primer binding upstream of PvanP and Rv primer downstream SfiI cloning sites.
Succesful transformation will have a size of 427 pb
?. [Clone sgRNA sequence into pYCR]
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
?. [Clone flanking regions into pYCR containing sgRNA]
Digest the pYCR(+sgRNA) and the repair fragments with SfiIInactivate SfiI by PCR clean up
?. [Clone flanking regions into pYCR containing sgRNA]
Ligate the components using T4 ligaseHeat inactivate the ligase (incubate )
60 °C
?. [Clone flanking regions into pYCR containing sgRNA]
Perform SmaI digestion to get rid of the original pYCR
?. [Clone flanking regions into pYCR containing sgRNA]
Transform the ligation mixture into E. coli MC1061 using the heat-shock methos. Grow the cells on LB agar plates + erythromycin
?. [Clone flanking regions into pYCR containing sgRNA]
To select the transformants perform a colony PCR with Fw primer binding upstream of PvanP and Rv primer downstream SfiI cloning sites.
?. [Clone flanking regions into pYCR containing sgRNA]
Miniprep and sequence the constructs
?. [Transform final construct in B. mycoides M2E_15]
Transform using an electroporation protocol using plasmid DNA
5 µl
{"blocks":[{"key":"5jor","text":"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. ","type":"align-left","depth":0,"inlineStyleRanges":[{"length":49,"offset":0,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"34qtr","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"5evts","text":"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.  ","type":"align-left","depth":0,"inlineStyleRanges":[{"length":21,"offset":0,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"4qbf1","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"cq2uf","text":"The plasmid pAD-KPS12-Pman will used as a starting point for introducing the antitoxin gene (ygcF) in B. mycoides. The vector will be cut with XbaI and SphI and ligated with the PCR amplified ygcF (using ygcF_Fw and ygcF_Rv as primers). In order to make the antitoxin expression inducible by solanine we plan on replacing the mannose inducible promoter with a promoter induced by solanine. To accomplish this, we will use the primers Psol_Fw and Psol_Rv to amplify the solanine promoter. The pAD-ygcFvector will be digested with EcoRI and XbaI, and ligated with the solanine promoter PCR product that will be digested as well with the up mentioned restriction enzymes. ","type":"align-left","depth":0,"inlineStyleRanges":[{"length":13,"offset":102,"style":"italic"}],"entityRanges":[],"data":[]},{"key":"6pds5","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"20h85","text":"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. ","type":"align-left","depth":0,"inlineStyleRanges":[{"length":5,"offset":242,"style":"italic"}],"entityRanges":[],"data":[]},{"key":"7n4q0","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"bhbo0","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Transform final construct in B. mycoides M2E_15]
Plate on LB agar containing erythromycinGrow at  overnight
28 °C
?. [Transform final construct in B. mycoides M2E_15]
Pick colonies and test them by colony PCR If the expected band size is present, purify the plasmid and sequence
?. [Transform final construct in B. mycoides M2E_15]
Grow the colonies with the expected sequence on liquid LB + erythromycin + manose Incubate
28 33
?. [Transform final construct in B. mycoides M2E_15]
Next day pick colonies in BHI medium (without antibiotic) and grow at overnight
37 °C
?. [Transform final construct in B. mycoides M2E_15]
Dilute the liquid cultures and plate them on LN (no antibiotic)grow overnight st
37 °C