From a364eb51c24fa8dc1c28abfb416244e1eb7cf65f Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Tue, 4 Mar 2025 14:45:23 +0100
Subject: [PATCH] mutant generation final steps

---
 .../isa.assay.xlsx                            | Bin 9257 -> 9258 bytes
 .../isa.assay.xlsx                            | Bin 11884 -> 11885 bytes
 assays/Metabolomics Analysis/isa.assay.xlsx   | Bin 10745 -> 10746 bytes
 assays/Mutant Generation/isa.assay.xlsx       | Bin 16547 -> 22970 bytes
 .../protocols/R401andEcoliConjugation.md      |   3 +++
 .../SucroseSecondHomologousRecombination.md   |   3 +++
 .../isa.assay.xlsx                            | Bin 11665 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12307 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15938 -> 15939 bytes
 10 files changed, 6 insertions(+)
 create mode 100644 assays/Mutant Generation/protocols/R401andEcoliConjugation.md
 create mode 100644 assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md

diff --git a/assays/BGC prediction using antiSMASH/isa.assay.xlsx b/assays/BGC prediction using antiSMASH/isa.assay.xlsx
index b06b8354d92b038d824c16bc5ed5745d5632e109..0c87e3146728ed2f1778c0a9d98276d12929de68 100644
GIT binary patch
delta 724
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diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
index e846156dd260e8768b142f4e0c7d8c94e6b7ff3c..13af4a103f05c97a1988a354cd472f29773efa4d 100644
GIT binary patch
delta 768
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diff --git a/assays/Metabolomics Analysis/isa.assay.xlsx b/assays/Metabolomics Analysis/isa.assay.xlsx
index c876c4ee3472462539cb9506cce354ad57473dfa..2828a44d204d086e4ac9ffa61342b8a760a97d20 100644
GIT binary patch
delta 721
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diff --git a/assays/Mutant Generation/isa.assay.xlsx b/assays/Mutant Generation/isa.assay.xlsx
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GIT binary patch
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diff --git a/assays/Mutant Generation/protocols/R401andEcoliConjugation.md b/assays/Mutant Generation/protocols/R401andEcoliConjugation.md
new file mode 100644
index 0000000..75c4ee8
--- /dev/null
+++ b/assays/Mutant Generation/protocols/R401andEcoliConjugation.md	
@@ -0,0 +1,3 @@
+## Conjugation of E. coli and R401 and selection for first homologous recombination event
+
+*E. coli* BW29427 cells containing the plasmid and R401 were inoculated into 4 ml of 50% TSB containing 25 μg/ml Kan and 50 μg/ml DAP or 50% TSB and incubated overnight at 37 °C with 180 rpm agitation or 25 °C with 180 rpm agitation, respectively. Cells were harvested by centrifugation at 8,000 rpm for 2 min at room temperature, then washed 3x and subsequently resuspended in 1 ml of 50% TSB followed by centrifugation, after which the supernatant was discarded. After quantifying OD600, both cultures were mixed to equal parts and approx. 10x concentrated by centrifugation. The bacterial suspension was plated on 50% TSA plates containing 50 μg/ml DAP and incubated at 25 °C overnight to allow for conjugation events. The mating patches were scraped of the plate and resuspended in 1 ml 50% TSB. Then, 100 μl were spread on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Nitrofurantoin (Nitro; Sigma-Aldrich; to counter-select *E. coli*) and incubated at 25 °C. Colonies were validated for successful genomic insertion of the plasmid via colony PCR using a primer specific to the genomic DNA approx. 150 bp upstream of the upward flanking region (upup) and the plasmid specific M13F primer. Colony PCR was performed on at least 15 separate colonies and a WT control with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously, but with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis followed by Sanger sequencing following the manufacturer's protocol.
\ No newline at end of file
diff --git a/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md
new file mode 100644
index 0000000..dcfd4b1
--- /dev/null
+++ b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md	
@@ -0,0 +1,3 @@
+## Sucrose counter-selection to induce the second homologous recombination event
+
+A R401 colony with a successful genomic insertion of the plasmid was resuspended from a plate into 1 ml of 50% TSB. The cell density in the medium was then measured using the Multisizer 4e Coulter Counter (Beckman Coulter) following the manufacturer's protocol. One hundred microliters of 500 cells/μl, 5,000 cells/μl and 50,000 cells/μl dilutions were spread on three separate 50% TSA plates containing 300 mM sucrose. The plates were incubated at 25 °C for approx. 48 h. At least 30 colonies were examined by colony PCR using the respective upup and dwdw primers. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol. They were also streaked on 50% TSA containing 25 μg/ml Kan to verify loss of the plasmid. A second colony PCR was performed on positive colonies and a wt control to validate the absence of the GOI, using a forward (inF) and reverse (inR) primer inside the GOI. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25 μl reactions as described previously. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification, 4 ml of 50% TSB were inoculated with a positive colony and grown overnight at 25 °C at 180 rpm. Finally, 750 μl of the overnight culture were added to 750 μl of 50% glycerol in an internally threaded 1.8 ml Nunc CryoTube, gently mixed, and stored at -80 °C.
\ No newline at end of file
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z;{c*S%8X^f%KT)(ruEBOLL^K<6r-FCQrx=9nSliB<YXA5C(o8sVtXM4OlAxKxn~W}

delta 628
zcmX?Nd)SsIz?+$ci-CcGgF(7-BTok-lXT_eX^cuhYV$5e0WkLrlRQZDEt4upRD@X`
zENaT41>z>Md|?LB;T&=-AXVi&U{&*YcC&$mqlM=)frS5vf`ygEwlH!^SB6Gq+z4-r
zoBTjryIxDARbi{>a-~qM(AD>1syX#Kb8mV(|J!%2nL%s$y|S{j<G%~G*@UHjV7}(L
zR!n7{iweWhtrJ#7OxS)_YtG~Yios1&qYgHiJUC&r|MU*eo!dSnZDs5hih0mG#d~Mu
z1ldKNnx!@DqQ%1N1LobyiZ=XmLF|gbzI4_LDqZg~Sx>OkJG(_6zR=T`vO%O~iwS#l
z8Cz4Z#zLmnzGIVT%-hPbt;n3)>X}odXAaMuDf3PQ)|M2BTr0I*xqR-G@A8-Xzkbd(
z&(a9pt;MHup2dGBYul}7`C02r)*4k!%&wdg;dF0l`vv!RAHMR+{QkmGqjR88oA;OX
z*C(Ne<|p&}u3cL&Z*Q#A+ocjt3v(|Ar$2E#DYNeH&h0`+oQvOm)t}0{`)xXd;E%aW
z{<8U>`TgZ%=4PczA=S+WUWZif9ocL$Yx(Az>9;?J{=K<DO77?O$0rkJynhw{Y(H|0
zZcdl%1;^GmDSJ>fm`fXhsBCF-5Vb^FA4Hv&m6`leS`x(Mk#Pf2Au?7VYJ!Xlh`J`@
y0HQ$3Ok}~z{AIzWO^~&ONSJ~sCOI3VsCAb!0}0m4$uLGuo+GEk_FM{>$`}CgyZM6v

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index f79bce8581a85dd69170620fa7fb175e0bed438c..725e55d80bc97a6a08547243fe24be57e8c0e833 100644
GIT binary patch
delta 761
zcmbQ7FeQN}z?+$ci-CcGgJDn2MxN!2OnYi3Z)H>hQk!ow3V^w<ndCvDiPEA%@kOaQ
z#rnv4HXAeRf>kB4Xn{2Ku~e{tXb#R>OdwrPxD7y*7|#c=DDP*m=wJSiAnpf&2_VIj
zn?%(>+*P7$K(d=7B-oij!imzVAZ~~BZZ;6@sx%+0`I!n>m{;{ABj=u)(5TlHr}^V2
z-&fPF_Y!SY*ebeQDO77kX#Tpr4c<x7-!4tqzy7mA!>N_q=gz(P(QeM!{#3y~4W&V=
zB|F^(IUNdhm)tscqG~ILyvKtHng_YoCb(r4q|CdYdS^*Ybj8+N4X&JeAKXqYy>sRS
z-^C?UtoAie+Bq>?!#{6}u4Gll_A3Va(xVr6irl>{G=a0et;eiwp`?swzUGgFOulUy
zye!KD95}gTd)*CfvjuZr=80`N)4FL&g!B$g+lkG;UOZ7)vnng3H+toJ`(^T{r*AL2
z60j;QRK{Z-=e#0ro~>uEXRR+;YE(5byJAX&!@Xs{FSx(^@Re6)_ZNm5odbp1yuYkV
zpM)NopUhvnbZx=By>U)wmr5K8yS;1ixf9JzmNCDI^Hn;I|GM)!Uh8%F?XwI@AFMrh
z^Uj;`{p01&S&pAnywnn>w0Q36Qh!-7%Wv6y-}B*L(+_W|e{#Q1y}54R>iU`gnNXrS
zRbvx4swK7TLD3VXWdx%7w9G-&aV>oiC8aMjSwveB#MIYz15xGLRv>D#whM^*38CzD
z96-EA9e)sYNe3(^p$q0^>VoA~>zaY&9z%GldSF42@y&W*<LBvtU2$K}5~NH@-xNdz
d>f0ctiD~*^BhKl|Fh)&&t*^w!qXkUr3;+&*Kra9Q

delta 771
zcmbP|FgbxIz?+$ci-CcGgF(7-BhPY1Ch5w_TN#yr)aILv0$}cICV7x(qO_<`d{Jsn
zu|Be%&Bn~SU{y&hS|Ck*EEOyunuGHe6G+z+ZUYb{#`6Iz%KI5C`j`JBi2Ffc0!Z=X
zCQ&sIca`WGknH9N33g_XaH6y-h}$8(n+-&}D$NILex?Ez=2bn($SGYJ8kKP)ye)3>
zeKqZREs<7*t)k17LbXCy-;1f{)a%T>>FxY)-??T6t>yR1%F>SiF4$%hmimGDn&(<E
zm3b~I3`e(4SQRm0`&q3ylMg5cH%*N?*ktnHgwg)fJ3M!8`;fGiv0EtSLGKjroskn{
z7kO%y*075f3$G8DcPA^_@XH0UD+c@0Sudz`y~|`h!BX$+7Jc|aPhZLgk(w<g?9pXx
zO~D!qnOggfO`b7tE627Xb8f3=PLZBDJa?wdI}uo0QY3P%)OO|axmUi+U+(|<Iomu-
zBXqYGpUQa_|DCLDx1Qx^tuI+?R5dZXa!Q2Ly`}9J+~0lp$}98x3rCI4fkJKGU)Eor
zgdUoo%<sE)ZNa>~u}*K7N;oady&RnW#POuey1zTO3mtJTe)m;>D(~*M=?sED<}Ufm
z=6~k*myem7l`4f)Hyd~zQn`0zv&pRGn{TGy{v7)E<_0ObpW7dwOqlWhRs6I4C^4O;
zu?Za0Qd;()*ooFM0#W^1<{;{XmOhA*)|Z(qsx1j(8fd$Ls0wW>5Vb|y1w{RVP!2i{
z;Hb;2nsH>~WCdN($@6rKLGqV%z{(_b{Xx7eT?Y`gM%N5PJ%RAl^uU53n_Be1HqF-q
pJLrL)B}kdHzA1<b(ziiM9n<x}Mx57|VT_#oMqi1ITML-z834|bC0qai

diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 36f4e75c4a78fa0fdeaa5b15460c17377974b9a2..0ea160aee9616d65c884afa9f8da53d84478e394 100644
GIT binary patch
delta 826
zcmX?9bGU{lz?+$ci-CcGgJDn2MxG6fOnYi3?_*Q~Qkx$!3NR|~sR@k&O2Yv&kX4bR
z57Wi~)HYd^Srw$-hS>zHy@*8%#GS`7hXpLmxdcQ{e#C14qD1&UfJE8&qnSBDhC5zv
zo3m-MG@r`kKA~eEnJ{5<W{|>uv5z2blejw@h<jRA4aEH{dkQ4GdB5TWFde8G23EaN
z1MG$i8oNQNH}~q#X9DTcGXe{T8l7bX+xEKRG=KbLZ4>Q!FVR+ot)k17LbX<e=C9k^
z;GGox?b3w(>pv?roLaek?%bOn?dF{APZj*rP#UyaveR9V)1gpz$*prIs<v{-dpww+
zd5~*uf?HNW%Dnrjcb3FNS8To2;L54@!R^%2J7-StU0gE7YG3oDofE?~{PVWxN>*iT
zzhba2J$iws$lc3A6FBSJdd%7uO3HZVYyL>c<lC0P%d$Mcfs;$N*WJ)ITQKKkp4gT%
zt(&GqNbk_Jo!I>A#S?`!tFl6RqgTGSUnYNg`u4Ib0jttNWjy9_&MV^P*?RVR*7}mA
zMpYBDE2cy^+*|hhg8RD<UwLJAe_^Q6IZ&w0`^&oYN$8>Z$^4~D*A~p%8|QR(sl=hM
z+q)K@JJH-^8S|?+U!~*tuRE{fwO*IsKFgr=!P;{-@4Ok`KVJTv<@ia(OD%Cqi|3v$
z^_La1{Fcr4Js<ux{qUyxC-?i*o9p(iuAljz36>^6Non&&(>>tCkZ*1eiiZv6Mj+~m
zxjBfEwa^Dqeio)6s@1{`M4h#;0#TfnE+EPuLQS=F0P(I{`hzG9E3jOi6_~dl!eg}t
z^TMpbDrZ=mfmB|A@Pur@f*}yf&q8MMCL2kR{8<~Yv-oT+K|GK|sx4Te!PW*Tp&YdZ
VoA%dMhH=?sDLW;$By&(s005(}R!jf@

delta 836
zcmX?HbEt+Vz?+$ci-CcGgF(7-BhLm#Ch5w_`xupg)aFNw0*uPim7!5UX*gg8vMO@)
zVcHmg+9r!KtAf<qFq?q27qMu8xbs-%uz-a*mw@QWk9ZA0lnCDkkSH5}G&3j2aL3DS
zb2d$u=2My6Cv*%X6DDlV3{u!H_7TKw5_e|<aZk&tfw-S#Pl04N?^m1vrUO;Oz^Yej
zfZcFGV>d|k=3f2zOdwr)MquGkqqB@)+cIv1x5Z7?Hqoxv5@}V~D!N=LR4a7#y_jlF
zz0Ta5-p>E_ooi;$T7IvrEbaL3f^9ZosUMiHd9D>xndhRyaCGa0RS^@mpVgW(`G8_@
z)6}SgO(qXc80|m3!*l1h4@p}YyM<yN^iJ{K8970Ak*8*94ZCQu@cMvxce0`lzg!Tz
zVz4ir^@2**yG+&-EcMQA(T6Ye^rdVNso7$}9$m)P6s)n3skQIe<Qem}a%?Ly=eBz0
z6zQ46b7#uD6M?lQMIzTqZC5Uzd*!?Q<^Hdqv(2+KLU(KNshnr=-^to`>sfx*`jWLq
zRTHx-r$jj2TiSlX{oRMJyfVMPaMb7=DAeZtW&QO@=%M+^{Jv|~7R=il>-2W1gww*@
z%fabS98b!u`@3_y&=Ke2cVG3V^6q|{&LH?>?vlT3{%3xF`Ixy`sZvOFvw_zkm3v1v
zo6K6i`DXg<&!K;BZjh4ux&86Ugc<K&#Xs8*OA?@zw0V>19&lPHFt-Oq!$xx>5cSmD
z97M@k=z}PK3sVr)X5j{+&RJN2C@xDE5ETHSrdc|GcsDHlL6oKyST5fR%sXJ^0*=zO
zZHr|$O}=j_I$6ou7^E!R8mwuiwHb(a5yBI;0SksgD1Qr?$(wB?LGtHpz%JyswFL1%
i5^1(ziAGx+q$G397Hrx-TN%cslcnvH*b>b_c>(|rwKxp`

-- 
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