TITLE: Cloning of Bacillus mycoides 
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><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><div class = "text-block"><div class = "justify" style = "text-align:left"></div></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"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>The plasmid pAD-KPS12-Pman will used as a starting point for introducing the antitoxin gene (ygcF) in </span><span style = "font-style:italic;">B. mycoides. </span><span>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. </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"><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"></div></div></div>

[STEPS]
?. [Making electro competent cells]
Pick 1 colony of B. mycoides M2E_15 and inoculate it in BHIS
?. [Making electro competent cells]
When OD600nm reached 0.85 add 2% glycine and 2% threonine in order to weaken the cell wall.
?. [Making electro competent cells]
Grow the cells overnight at
30 33
?. [Making electro competent cells]
Dilute the overnight culture 50 times in LBSP medium until the OD600nm reaches 0.65
?. [Making electro competent cells]
Collect the cells by  . Discard supernatant
Centrifuge: 4000 34, 10 min, 4 10
?. [Making electro competent cells]
Wash cell pellet with pre-chilled electroporation buffer (10% glycerol, 0.25 M sorbitol, 0.25M trehalose) x4
?. [Making electro competent cells]
Suspended in 1 ml electroporation buffer (10% glycerol, 0.25 M sorbitol, 0.25M trehalose).
?. [Making electro competent cells]
Snap-freeze the electrocompetent cells in liquid nitrogen and sore at
-80 °C
?. [PCR -  Phusion cloning]
For the PCR mix pipette: -  primers mixture Fw + Rv ( 5 uM) (see table ''Primers'')-  HF buffer-  dNTP (10mM)- DNA template (0.1 ng)- phusion polymerase- MIliQ to
5 µl
10 µl
1 µl
0.25 µl
50 µl
?. [PCR -  Phusion cloning]
PCR reaction is performed with the following protocol:  AB1   Temperature (*C)      Time (mm:ss)   2  98    05:00  3  98    10:00  4  Variable    00:30  57200:306repeat above steps30x77210:008  11    Infinite
AB1   Temperature (*C)      Time (mm:ss)   2  98    05:00  3  98    10:00  4  Variable    00:30  57200:306repeat above steps30x77210:008  11    Infinite
?. [PCR -  Phusion cloning]
Load  of sample plus   of staining solution 6x. Check the length of the band (bp).Purify sample : PCR cleanup/ gel extraction according to manufacturer.
5 µl
1 µl
?. [Cloning ]
Digest the polymerized insert and the desired vector with 2U of apropriate restriction enzyme. See table ''Primers'' in order to choose the enzyme.
?. [Cloning ]
Digestion mixture ( ):-SfiI 2 U- Plasmid ( )-Insert ( )-  CutSmart 10x (or appropriate buffer)-MiliQ to
20 µl
50 ng
20 ng
2 µl
20 µl
?. [Cloning ]
Incubate   at the temperature recommended by the manufacturer of the restriction enzyme
?. [Cloning ]
Inactivate the restriction enzyme by incubationg 20 min **SfiI can't be heat inactivated so in order to inactivate it a PCR clean up is necesary
65 °C
?. [Cloning ]
Ligate  the digested insert and vector.Ligation mixture : -10 ul digestion product -1 ul T4 -2 ul T4 ligase buffer-7 ul MQ
?. [Cloning ]
Incubate
0 Room temperature
?. [Transformation (electroporation)]
Thaw on ice of electrocompetent cells
100 µl
?. [Transformation (electroporation)]
Add   of plasmid and the aliquot of electrocompetent cells to ice-cold electroporation cuvettes
2 µg
?. [Transformation (electroporation)]
Electroporate 25 uF, 10 kV/cm, 200 Ohms
?. [Transformation (electroporation)]
Add 1 ml of BHIS and incubate  for recovery
30 33
?. [Transformation (electroporation)]
Plate on LB + Agar (1.5%) + 100 μg/ml spectinomycin.For crispr cloning, to activate cas9 expression, add 0.2% mannose.
?. [Mutant selection]
Incubate at
30 °C
?. [Mutant selection]
Randomly pick potential mutants and test by colony PCR (for primers use the ones that flank the region of insertion sites).
?. [Mutant selection]
After selecting colonies that show the expected band size, purify the desired plasmid (miniprep) and transform into B. mycoides.