DTU igem15 e-lab notebook: Oligo competent strains (OCS)

Transcription

1 DTU igem15 e-lab ntebk: Olig cmpetent strains (OCS) Cntent Cntent... 1 muts::gp35 ne R and muts::beta ne R... 2 Re d f Amplificatin f the muts up and dwnstream fragments: : Re d f Amplificatin f the gp35 ne and the lambda beta ne cassettes: : Gibsn Assembly f mutsgp35 ne cassettes and muts lambda beta ne cassettes with redbibrick psb1c3 (psb1c3_muts:: GP35ne R ): Transfrmatin f psb1c3_ muts::gp35_ne R and psb1c3_ muts::beta ne R : Clny PCR f muts::gp35 and muts::lambda beta mutants: amye::gp35 ne R and amye::beta ne R PCR f pdg268ne backbne fr gibsn assembly f pdg268ne_lambda beta: Gibsn assembly f lambda beta Gblcks int pdg268ne and transfrmatin int E. cli: Linearizatin f pdg268ne_lambda beta: Gibsm assembly f gp35 int pdg268ne Preparing Natural cmpetent B. subtilis W168: Miniprep f lambda beta and gp35: Linearizatin f pdg268ne_gp35: Transfrmatin f B. subtilis with pdg268ne::lambda beta: Digestin 2 f pdg268ne_gp35: Transfrmatin f pdg268ne_lambda beta: Red 2: Clny PCR f gp35::amye:

2 muts::gp35-ne R and muts::beta-ne R Re d f Amplificatin f the muts up and dwnstream fragments: : Prefrmed by Viktr, Authr f e-lab ntes: Viktr Purpse: T amplify the fragments frm the B. subtilis genme. The tw fragments that will be amplified is muts upstream and muts dwnstream. After PCR was dne the prduct was purified. Prtcl N specific prtcl. Purificatin was dne using the QIAgen PCR purificatin kit. Materials: NEB phusin plymerase dntps 5x HF buffer Primers fr amplificatin f the upstream fragment primuts GP35+beta N fwd primuts GP35+beta N rev Primers fr amplificatin f the dwnstream fragment primuts GP35&beta C F primuts GP35+beta C rev Bacillus subtilis WT Sterile water Prcedure: 1. One clny B. subtilis was inculcated in 10 ul sterile water and biled fr 10 min. at 98 degc. 1 ul was used as template. 2. Duplicates was made 3. Master mix fr PCR reactin cntaining dntps and HF buffer PCR mix Sample Cncentratin (um) Vlume (ul) Cmments NEB phusin 0.5

3 plymerase dntps 10,000 (10 mm) 1 5x HF buffer 10 Fwd primer 25 1 Rev primer 25 1 Template (biled cells) 1 H2O 11 Ttal 50 PCR tubes Tube Template Fwd primer Rev primer Cmments Biled B. sub. WT primuts GP35+beta N fwd primuts GP35+beta N fwd primuts GP35&beta C F primuts GP35&beta C F primuts GP35+beta N rev primuts GP35+beta N rev primuts GP35+beta C rev primuts GP35+beta C rev Amplifying the upstream fragment Amplifying the dwnstream fragment PCR prgram Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin Final extensin 72 2:00 Hld 4 Infinite

4 Data: lane 1 Ladder lane 2 cpcr sample 1 lane 3 cpcr sample 2 lane 4 cpcr sample 3 lane 5 cpcr sample 4 lane 6 lane 7 lane 8 lane 9 lane 10 lane 11 cpcr Bacillus WT cpcr plasmid pdg268::gp35 N sample laded mutsl up mutsl up mutsl dw

5 lane 12 mutsl dw The purified PCR was nandrped and fllwing averages was calculated: Purified muts upstream: 31 ng/ul Purified muts dwnstream 3: 29 ng/ul Purified muts dwnstream 4: 50 ng/ul Purified gp35: 62 ng/ul Purified lambda beta: 38 ng/ul Results and Cnclusin: Cells were lysed and band n abut 500 bp, which is what we expected. There purificatin was

6 Re d f Amplificatin f the gp35-ne and the lambda-beta-ne cassettes: : Prefrmed by Viktr, Authr f e-lab ntes: Viktr Purpse: Since the there was n result the first time we will d almst the same. After the PCR was dne it was purified. Prtcl N specific prtcl. Purificatin: QIAgen PCR purificatin kit Materials: NEB phusin plymerase dntps 5x HF buffer Primers fr amplificatin f the gp35 ne and lambda beta ne cassettes primuts GP35+beta muts fwd primuts GP35&beta muts R Template: gp35_pdg268ne and lambda beta_pdg268ne Sterile water Prcedure: 1. Master mix fr PCR reactin cntaining dntps and HF buffer PCR mix Sample Cncentratin (um) Vlume (ul) Cmments NEB phusin plymerase 0.5 dntps 10,000 (10 mm) 1 5x HF buffer 10 Fwd primer 25 1 Rev primer 25 1 Template N/A H2O 11 Ttal r 53 PCR tubes Tube Template Fwd primer Rev primer Cmments

7 1 pdg268ne_gp35 2 primuts GP35&beta muts R primuts GP35&beta muts R pdg268ne_lambdabeta primuts GP35+beta mutsfwd primuts GP35+beta mutsfwd 2 ul f template was used 3 ul f template was used PCR prgram Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin Final extensin 72 2:00 Hld 4 Infinite 4. On the gel bands at abut 6000 bp was bserved, this culd be plasmid leftvers. T remve this the PCR reactin was digested with DpnI restrictin enzyme. a. 0.5 ul DpnI was added t each PCR reactin b. Tubes were flicked a cuple f time and centrifuged fr 2 sec. c. Then incubated fr 50 min at 37 degc 5. The reactins was then purified using QIAgen PCR purificatin kit.

8 Data: Lane 1 Lane 2 13 Lane 14 Lane 15 Ladder Nt fr this experiment GP35 lambda beta The purified PCR was nandrped and fllwing averages was calculated: Purified gp35: 62 ng/ul Purified lambda beta: 38 ng/ul Results and Cnclusin: The PCR was successful 2 bands f abut 2700 bp was bserved at the gel. The PCR prduct purified.

9 Gibsn Assembly f mutsgp35 ne cassettes and muts lambda-beta ne cassettes with redbibrick psb1c3 (psb1c3_muts:: GP35ne R ): Prefrmed by Karlina, Aurthr f e-lab ntebk: Karlina Purpse T assemble the lambda beta_mutsko and the gp35_mutsko int psb1c3 plasmid. If the experiment is successful the prduct will lk as this: The gp35 versin is the exact same plasmid just the CDS f lambda beta is changed fr the CDS f gp35. Prtcl NEB Gibsn assembly prtcl. Materials Mut dw3 (50ng/ul) GP35 (90ng/ul)

10 Mut up (31ng/ul) psb1c3 (red bibrick A) (79,3ng/ul) H2O Gibsn Master Mix Prcedure Tw different gibsn reactins was setup: Vlume (ul) GP35 1,8 Mut dw3 0,7 Mut up 1,1 psb1c3 1,5 water 4,9 Master mix 10 Vlume (ul) Lambda beta 4,5 Mut dw3 0,7 Mut up 1,1 psb1c3 1,5 water 2,3 Master mix 10 Gibsn Assembly run n Thermcycler fr 1h at 50 C. Chemical transfrmatin, cells with different transfrmats were plated n Cam 6y and ne 5 y. Verificatin f plasmids One O/N culture was made frm each plate. Samples were miniprept and linearized using XhI.

11 Data Plate Number f transfmants muts_gp35 (5y ne) 216 muts_gp35 (6y cam) 210 muts_lambda beta (5y ne) 90 muts_ Lambda beta (6y cam) 120 Digestin results

12 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 lambda beta lambda beta GP35 GP35 ladder

13 Only a small amunt f plasmid gt digested. The expected bands was at 892 bp and 4823 bp fr psb1c3_ muts::beta ne R. The difference in intensity f the bands may be because the large DNA fragments binds mre ETBr e.i. they are mre intense even if they are the same cncentratin. Cnclusin Transfrmatin was successful and plasmid was verified and linearized by restrictin digestin.

14 Transfrmatin f psb1c3_ muts::gp35-ne R and psb1c3_ muts::beta-ne R : Prefrmed by Pernile and Viktr, Authr f e-lab ntes: Viktr Purpse: A successful transfrmatin f the tw cassttes will result in tw different B. subtilis 168 strains: 1. muts::beta ne R 2. muts::gp35_ne R Prtcl Cfrench:BacTrans2 Materials: Natural cmpetent B. subtilis W168 frm Preparing Natural cmpetent B. subtilis W168: Plates: LB + nemycin 5y Template Linearized psb1c3_ muts::gp35_ne R and psb1c3_ muts::beta ne R Prcedure: 1. Prtcl was fllwed Transfrmatins Tube DNA Vlume (ul) 1 Lambda beta (1) 30 2 Lambda beta (2) 30 3 Gp35 (1) 30 4 Gp35 (2) 30 5 (negative cntrl) N N Plates Plate Antibitic Sample (tube) Vlume f sample (ul) Ne 5y (negative cntrl) Plates was incubated at 37 degc O/N

15 Data: Plate Number f transfrmants (Negative cntrl) 1 Results and Cnclusin: The clnies will be tested using a cpcr, even thugh there is a clny n the negative cntrl.

16 Clny PCR f muts::gp35 and muts::lambda-beta mutants: Prefrmed by Viktr, Authr f e-lab ntes: Viktr Purpse: T verify whether the gp35 r lambda beta gt inserted int muts in the genme f B. subtilis. These are transfrmants by linearized psb1c3_muts_lambda beta_ko r psb1c3_muts_lambda beta_ko. Fur different transfrmant are examined (see materials). Prtcl NEB HF phusin plymerase Materials: NEB HF Phusin plymerase NEB HF phusin buffer NEB dntps Primers: pdg268ne lambda cpcr rev primuts GP35&beta muts R Transfrmants (frm Transfrmatin f psb1c3_muts::gp35_ne R and psb1c3_muts::lambdabeta_ner: ) Gp35 (1) Gp35 (2) Lambda beta (2) Negative cntrl Prcedure: 1. A clny was inculated in 10 ul ddh2o. 2. Cells were biled fr 10 min. at 95 degc. 3. The PCR tubes was mixed as stated belw. PCR mix Sample Cncentratin (um) Vlume (ul) Cmments NEB phusin plymerase HF buffer 5x Plymerase was added after initial denaturatin

17 dntps 0.4 Fwd primer Rev primer Template (biled cells) 1 H2O 13.6 Ttal 20 PCR tubes Tube Template Fwd primer Rev primer 1 (psitive cntrl) WT B. subtilis 168 primuts GP35+beta Nfwd Expected band size (bp) primuts GP35+beta N rev gp35 (1) pribibrick_gp35_beta_r Gp35 (2) Lambda beta (2) pribibrick_lambda_beta_r (Negative cntrl)** WT pribibrick_gp35_beta_r N band 6 (negative cntrl)** WT pribibrick_lambda_beta_r N band PCR prgram fr tube 1 Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin Final extensin 72 5:00 Hld 4 Infinite PCR prgram fr tube 2, 3 and 5 Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin Final extensin 72 5:00 Hld 4 Infinite

18 PCR prgram fr tube 4 and 6 Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin Final extensin 72 5:00 Hld 4 Infinite Data: Lane 1 Lane 2 Ladder Psitive cntrl Lane 3 Gp35 (1) Lane 4 Gp35 (2) Lane 5 Lambda beta (2)

19 Lane 6 Lane 7 Negative cntrl (gp35) Negative cntrl (Lambda beta) The psitive cntrl is really faint, this might be due t that nly a small a amunt f WT cells was inculated in the water. The band is thugh the right size. Lane 6 and 7 are as expected with n bands. Results and Cnclusin: KO by insert f a recmbinase was cnfirmed in all clnies.

20 amye::gp35-ne R and amye::beta-ne R PCR f pdg268ne backbne fr gibsn assembly f pdg268ne_lambda-beta: Prefrmed by Pernille, Authr f e lab ntes: Viktr Purpse: Amplify the pdg268ne plasmid using primers that amplify the whle plasmids, but the lacz gene. In this way we will later be able t insert the recmbinases int the plamid. We expect a band n 6348 bp. Figur 1: pdg268ne. Marked is the amplified part. Prtcl NEB HF Q5 plymerase 2x master mix prtcl QIAgen PCR purificatin kit prtcl

21 Materials: NEB HF Q5 plymerase 2x master mix Primers pdg268ne rev gibsn pdg268ne fwd gibsn Template: pdg268ne dh2o QIAgen PCR purificatin kit Prcedure: 1. Prtcl was fllwed and PCRs was mixed as belw: PCR mix Sample Cncentratin (um) Vlume (ul) Cmments Q5 plymerase mastermix 25 Fwd primer 25 1 Rev primer 25 1 Template (pdg268ne) 1 H2O 22 Ttal 50 PCR prgram Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin Denaturatin Annealing cycles Elngatin 72 2:10 Final extensin 72 2:00 Hld 4 Infinite PCR purificatin 2. After the length f the fragments was verified n a agarsegel the PCR was purified using the QIAgen PCR purificatin kit.

22 Data: LANE 1 2 Lg DNA Ladder kb LANE 2 mastermix 1 LANE 3 mastermix 2 LANE 4 mastermix 3 LANE 5 LANE 6 Blank 2 Lg DNA Ladder kb Results and cnclusin Amplificatin was successful, because bands at just abve 6000 bp was bserved n the gel. Prduct was purified.

23 Gibsn assembly f lambda-beta Gblcks int pdg268ne and transfrmatin int E. cli: Prefrmed by Pernille, Authr f e-lab ntes: Viktr Purpse: T insert the tw lambda beta Gblcks int the linearized (by PCR) pdg268ne frm PCR f pdg268ne backbne fr gibsn assembly f pdg268ne_lambda beta: And amplify the plasmid using E. cli DH5 alpha cells. Figur 2: The lambda beta cassette was fused using tw Gblcks that was verlapping, but t make the simulatin easier the fused versin f the Gblcks is used. Fragment.FOR and Fragment.REV primer are imaginary primers nly used in the simulatin.

24 Prtcl NEB Gibsm assembly kit prtcl. Materials: #3 purified PCR (pdg268ne) Gblck lambda beta up Gblck lambda beta dw NEB gibsm asssembly kit Sterile water NEB E. cli heatshck cmpetent cells (c2887) Prcedure: 1. Fllwing was mixed in a PCR tube Gibsn assembly mix Sample Cncentratin (um) Vlume (ul) #3PCR (pdg268ne) 80 ng/ul 1 Gblck lambda beta up 10 ng/ul 1.2 Gblck lambda betadw 10 ng/ul 1.6 Gibsn mastermix 10 H2O 6.2 Ttal This was incubated fr 15 min. at 50 degc in a thermcycler (as prtcl states). Transfrmatin int E. cli 3. 2 ul f assembly reactin was added t 50 ul NEB cmp. cells 4. Cled n ice fr 30 min. 5. Heated at 42 degc fr 30s 6. On ice fr 2 min ul SOC medium (frm NEB) was added 8. Incubated fr 60 min. in a small reactin tube shaking at 210 rpm and 100 ul was plated n each f fllwing plates: LB, LB + 50y Amp and LB + 100y Amp. 10. Incubated O/N at 37 degc

25 Data: Plate Number f transfrmants 100y amp (200 ul plated) y amp (100 ul plated) 8 50y amp (200 ul plated) 21 50y amp (100 ul plated) 0 Results and Cnclusin: The gibsn assembly and transfrmatin seems t by successful, but this will be verified later restrictin digestin.

26 Linearizatin f pdg268ne_lambda-beta: Prefrmed by Sct and Verenat, Authr f e-lab ntes: Viktr Purpse: Linearize and verify the length f pdg268ne_lambda beta, which shuld later be transfrmed int B. subtilis. The reasn fr linearizatin is that the transfrmatin efficiency in B. subtilis will increase if the plasmid is linearized. Prtcl igem restrictin digestin prtcl Materials: Miniprep f gp35_pdg268ne frm Miniprep f lambda beta and gp35: NEB buffer 2.1 Sterile water Restrictin enzymes XcmI PvuII Prcedure: Tube 1 Restrictin digestin mix (50 ul reactins) DNA Cncentratin Vlume f Restrictin f DNA (ng/ul) DNA (ul) enzyme Lambdabeta # Buffer Water (ul) 21.3 Predicted bands 2 Lambdabeta # ng/ul Lambdabeta #9 and 1 ul buffer ul XcmI 5 ul NEB Lambdabeta # PvuII Lambdabeta # Lambdabeta # ul f each restrictin enzyme and 5 ul f buffer was used in each tube. 4.1 kb and 3.3 kb 1. The tubes was mixed as listed abve (n ice). 2. Tubes was flicked a cuple f time and spun fr 2 sec. 3. Incubated at 37 degc fr 1 hur

27 4. Samples was purified using the QIAgen PCR purificatin Data: Lane 1 Transfrmant 7 Lane 2 Transfrmant 8 Lane 3 Transfrmant 9 Lane 4 Transfrmant 10 Lane 5 Transfrmant 11 Lane 6 Transfrmant 12 Lane 7 Lane 8 Lane 9 Plasmid pdg268ne uncut plasmid Ladder Results and Cnclusin: The bands were as expected fr all digestins. Thereby we have verified that we d have the right insert.

28 Gibsn assembly f gp35 int pdg268ne and transfrmatin int E. cli: Gibsm assembly f gp35 int pdg268ne Prefrmed by Niclai, Viktr and Vilhelm, Authr f e-lab ntes: Viktr Purpse: Substitute the lacz gene in pdg268ne with gp35. Thereby make a plasmid gp35_pdg268ne that is able t insert gp35 int the amye lcus in the genme f B. subtilis.

29 Figur 3: The lambda beta cassette was fused using tw Gblcks that was verlapping, but t make the simulatin easier the fused versin f the Gblcks is used. Fragment.FOR and Fragment.REV primer are imaginary primers nly used in the simulatin.

30 Prtcl NEB Gibsm assembly kit prtcl. Materials: #3 purified PCR (pdg268ne) Gblck gp35 up Gblck gp35 dwn NEB gibsm asssembly kit Sterile water NEB E. cli heatshck cmpetent cells (c2887) Prcedure: 11. Fllwing was mixed in a PCR tube Gibsn assembly mix Sample Cncentratin (um) Vlume (ul) #3PCR (pdg268ne) 1 Gblck gp35 up 10 ng/ul 1.2 Gblck gp35 dw 10 ng/ul 1.6 Gibsn mastermix 10 H2O 6.2 Ttal This was incubated fr 15 min. at 50 degc in a thermcycler (as prtcl states). Transfrmatin int E. cli ul f assembly reactin was added t 50 ul NEB cmp. cells 14. Errr: cells was incubated fr 30s at 42 degc 15. Cled n ice fr 30 min. 16. Heated at 42 degc fr 30s 17. On ice fr 2 min ul SOC medium (frm NEB) was added 19. Incubated fr 60 min. in a small reactin tube shaking at 210 rpm ul was plated n each f fllwing plates: LB, LB 50y Amp and LB + 100y Amp. 21. Incubated O/N at 37 degc

31 Miniprep f gp35_pdg268ne (Date ) 22. Quick O/N cultures was made inculating ne clny in 3 ml LB + 50y Amp. 23. After 5 hurs f incubatin the quick O/N was miniprepped, but gt lw yield 24. New O/N cultures was inculated in LB + 50y Amp at 37 degc shacking at 200 rpm. Strage f O/N culture cells (Date ) 25. O/N frm step 14 was centrifuged fr 15 min. at 5000 rpm. 26. Supernatant was discarded and pellet was stres in 20 degc freezer (fr later miniprepping). Data: Date: transfrmant n 100y Amp plate and 14 transfrmants n 50y Amp plate. Results and Cnclusin: Transfrmatin went OK in spite that the cells was heat shcked twice. The pr yield in the first miniprep (step 13) was prperly due t the shrt incubatin time f the quick O/N culture. The cells might nt have prduced many plasmids.

32 Preparing Natural cmpetent B. subtilis W168: Prefrmed by Viktr, Authr f e-lab ntes: Viktr Purpse: Prepare natural cmpetent B. subtilis, t use fr transfrmatins. Prtcl Natural cmpetent Bacillus subtilis 168 Materials: One clny f B. subtilis W168 Minimal grwth medium (MGM) (fr recipe see prtcl) Starvatin medium (SM) (fr recipe see prtcl) 50% glycerl (t make glycerl stck) Prcedure: Duplicate was made f all the fllwing O/N culture in MGM (Date ) 1. One clny B. sub. was inculated in 10 ml MGM in a 250 ml Erlenmeyer flask. Incubated at 37 degc shaking at 180 rpm Making the natural cmp. cells (Date ) ml N/O (frm step 1) was inculated in 10 ml in a 250 ml Erlenmeyer flask. Incubated at 37 degc shaking at 180 rpm 3. After 2. 5 hurs (30 min. t early) 11 ml starvatin medium was added. 4. Cntinue incubatin fr 2 h 45 min. Glycerl stck 5. Eppendrf tubes were made cntaining (enugh fr 4 transfrmatins): a. 1.2 ml cmp. cells b. 264 ul 50% glycerl 6. These were stred at 80 degc Data Cells was nt tested yet Result and cnclusin N cnclusin since cells was nt tested.

33 Miniprep f lambda-beta and gp35: Prefrmed by Maja, Thea and Viktr, Authr f e-lab ntes: Viktr Purpse: T miniprep tw different plasmids frm E. cli. The plasmids which was minipreped was gp35_pdg268ne (fr insertin f gp35 int B. subtilis) and lambda beta_psb1c3 (T be submitted as a BiBrick). Prtcl QIAgen spin miniprep kit Materials: E. cli transfrmed with gp35_pdg268ne E. cli transfrmed with lambda beta_psb1c3 Buffers Buffer P1 Buffer P2 Buffer N3 Buffer PB Buffer PE Spin clumns Prcedure: 2. Prtcl was fllwed with fllwing exceptin a. Step 7 (nly recmmended) was dne. b. When prtcl stated centrifuge fr 30 60s centrifugatin was fr 60s c. Sterile water was used fr elutin 3. In ttal 12 (6 f each plasmid) minipreps was dne. Data: Cncentratin data generated by Nandrp (nly shwing the nce with high enugh cncentratin): Lambda 4: 21.5 ug/ul Lambda 5: 27.0 ug/ul gp35 1: 18.7 ug/ul gp35 2: 57.7 ug/ul gp35 4: 19.5 ug/ul

34 gp35 5: 23.1 ug/ul Results and Cnclusin: The minipreps was nt that efficient, but shuld gd enugh fr transfrmatin and verificatin.

35 Linearizatin f pdg268ne_gp35: Prefrmed by Maja, Niclai and Viktr, Authr f e-lab ntes: Viktr Purpse: T linearize gp35_pdg268ne, which shuld later be transfrmed int B. subtilis. The reasn fr linearizatin is that the transfrmatin efficiency in B. subtilis will increase if the plasmid is linearized. Prtcl N specific prtcl used. Materials: Miniprep f gp35_pdg268ne frm Miniprep f lambda beta and gp35: NEB buffer 2.1 Sterile water Restrictin enzymes XhI PvuII Prcedure: Restrictin digestin mix Tube DNA Cncentratin f DNA (ng/ul) Vlume f DNA (ul) 1 gp Restrictin enzyme Buffer Water (ul) gp35 2 gp XhI and PvuII NEB buffer gp ul f each restrictin enzyme and 5 ul f buffer was used in each tube. 5. The tubes was mixed as listed abve (n ice). 6. Tubes was flicked a cuple f time and spun fr 2 sec. 7. Incubated at 37 degc fr 1 hur 8. Samples was purified using the QIAgen PCR purificatin (date ). Data: Samples were nt nandrpped r run n a gel, since a verificatin digestin f the same samples was run parallel. Results and Cnclusin: N results fr this experiment.

36 Transfrmatin f B. subtilis with pdg268ne::lambda-beta: Prefrmed by Vilhelm, Maja and Viktr, Authr f e-lab ntes: Viktr Purpse: Tranfrm the pdg268ne_lambda beta int B. subtilis this will d duble hmlgus crss ver int amye in the genme and thereby the strain will express the lambda beta recmbinase. Prtcl Cfrench:BacTrans2 Materials: Natural cmpetent cells Linearized pdg268ne_lambda beta LB medium 5y nemycin + LB plates Prcedure: 1. Prtcl was fllwed with n exceptins and 100 ul was plated n 5y ne. 2. Plates were incubated at 37 degc ver night. Results and Cnclusin: Transfrmants was bserved and is t be verified by a clny PCR later.

37 Digestin 2 f pdg268ne_gp35: Prefrmed by Vilhelm and Viktr, Authr f e-lab ntes: Viktr Purpse: Since the first digestin f pdg268ne_gp35 did nt wrk, we will try again this time using a different restrictin enzyme. Prtcl igem prtcl: Prtcls/restrictin digest Materials: EcRV NEB buffer 3.1 pdg268ne_gp35 Prcedure: 1. Fllwing reactin was set up. All with a ttal vlume f 10 ul. Restrictin digestin mix Tube DNA Cncentratin f DNA (ng/ul) Vlume f DNA (ul) 1 gp Restrictin enzyme Buffer Water (ul) 2 gp ul 1 ul NEB gp EcRV* buffer gp pdg268ne N/A 2.5 N enzyme N buffer Tubes were flicked and centrifuged fr 2 sec. 3. Incubated at 37 degc fr 25 min.

38 Data: Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Ladder pdg268ne nt digested (cntrl) pdg268ne_gp35 #1 nt digested (cntrl) (Failed lading) pdg268ne_gp35 #1 nt digested (cntrl) pdg268ne_gp35 #2 nt digested (cntrl) pdg268ne_gp35 #3 nt digested (cntrl) pdg268ne_gp35 #4 nt digested (cntrl) Empty pdg268ne (cntrl) Lane 10 pdg268ne_gp35 #1 Lane 11 pdg268ne_gp35 #2 Lane 12 pdg268ne_gp35 #3 Lane 13 pdg268ne_gp35 #4 Results and Cnclusin: Gp35 1 and gp35 2 have the insert crrectly. This will be send fr sequencing.

39 Transfrmatin f pdg268 gp35 int natural cmpetent B.subtilis 168: Prefrmed by Verena, Authr f e-lab ntes: Verena Purpse: The DNA frm sample 1 (97.97 ng/µl) and sample 2 ( ng/µl) were transfrmed int natural cmpetent B.subtilis 168 and sent t sequencing at the same time s that transfrmants with cnfirmed crrect sequence are btained. The sequence is cnfirmed by sequencing and the presence f the plasmid in the single clny is cnfirmed by ding a clny PCR. Materials: List ut everything yu need t perfrm this experiment including lab equipment, media, enzymes, buffers etc. Plasmid DNA: 1 : pdg268 gp35 (97.97 ng/µl) 2 : pdg268 gp35 ( ng/µl) Linearizatin f Plasmid: Restrictin enzyme XhI (frm Jan s freezer) Restrictin enzyme PvuII (frm Jan s freezer) NEB buffer 2.1. Sequencing Primers: pribibrick Lambda Beta reverse pdg268 ne Lambda cpcr frward Prcedure: Transfrmatin: 1. Linearizatin f plasmid accrding t the manufacturer s prtcl. Incubatin fr 60 min at 37 C Sample Cncentratin (ug/ml) Vlume (ul) NEB Buffer µl XhI 1 µl PvuII 1 µl Plasmid DNA final: arund 1 µg 10 µl H2O 32 µl Ttal 50µL Table 1: Linearizatin f plasmid by using restrictin enzymes.

40 Transfrmatin int B.Subtilis accrding t the prtcl Cfrench:BacTrans2 200 µl frm each ube were plated in 5 γ ne plates. Plates were incubated vernight at 37 C Results and Cnclusin: 84 clnies grew n plate 1. n clnies grew n plate 2. Bth plates were stred in the freezer at 4 C. Transfrmatin f pdg268ne_lambda-beta: Prefrmed by Viktr, Vilhelm and Maja, Authr f e-lab ntes: Viktr Purpse: Transfrm the pdg268ne_lambda beta int B. subtilis 168. mutsl. pdg268ne:lambda beta is an integrative plasmid that is able t integrate int the amye gene f B. subtilis 168, thereby the strain will express the beta prtein frm the E. cli phage lambda red. Prtcl Cfrench:BacTrans2 Materials: Natural cmpetent B. subtilis 168 cells (frm Preparing Natural cmpetent B. subtilis W168: ) Three different samples with pdg268ne_lambda beta Sample #7 (46 ng/ul), #8 (56 ng/ul) and #12 ( 76 ng/ul) Prcedure: Prtcl was fllwed with fllwing exceptins: Fr pdg268ne_lambda beta we added abut 300 ng f each fragment. Negative cntrl with n DNA added was made pdg268ne_lambda beta transfrmants were plated n 5y Cam incubated O/N at 37 degc Data Fllwing transfrmants was bserved:

41 #7: 60 transfrmants #8: 20 transfrmants #12: 26 transfrmants Cntrl (n 5y cam): 0 transfrmants Results and Cnclusin: Transfrmatin f pdg268ne_lambda beta was successful and there will be made a glycerl stck f these.

42 Red 2: Clny PCR f gp35::amye: Prefrmed by Viktr, Authr f e-lab ntes: Viktr Purpse: T verify whether the gp35 gt inserted int amye f B. subtilisʹ chrmsme. This is a 3nd try f Clny PCR f psb1c3_muts_lambda beta_ko f muts_lambda beta KO and gp35::amye: Prtcl NEB HF phusin plymerase Materials: NEB HF Phusin plymerase NEB HF phusin buffer NEB dntps Primers: primuts GP35+beta C fwd primuts GP35+beta C rev pdg268ne lambda cpcr fwd gp35_seq_rev Template Transfrmants #2, #3 and #4 frm plate 1 frm Transfrmatin f muts_lambda beta KO and pdg268ne_gp35: WT B. subtilis 168 Prcedure: 4. A clny was inculated in 10 ul ddh2o. Fr ne clny f #2, #3, #4 and a WT B. subtilis After inculatin in the PCR tubes the clny was inculated in abut 3 ml LB + 5y nemycin, this was incubated at 37 degc O/N 6. Cells were biled fr 10 min. at 98 degc. 7. The PCR tubes was mixed as stated belw. PCR mix Sample Cncentratin (um) Vlume (ul) Cmments NEB phusin 0.2 Plymerase was added

43 plymerase HF buffer 5x 4 dntps 0.4 Fwd primer Rev primer Template (biled cells) 1 H2O 13.6 Ttal 20 after initial denaturatin PCR tubes Tube Template Fwd primer Rev primer Expected band size (bp) 1 (psitive cntrl) WT B. subtilis amye::gp35 ne R #2 3 amye::gp35 ne R #3 primuts GP35+beta Cfwd primuts GP35+beta C rev 591 pdg268ne gp35_seq_rev 1347 lambda cpcr fwd 4 amye::gp35 ne R #4 5 (Negative WT B. subtilis cntrl) 168 *Primer was named wrng when rdered. N band PCR prgram Step Temperature (degc) Time (hh:mm:ss) Cycle Initial denaturatin 98 10:00 Adding plymerase 4 Hld Denaturatin Annealing 56* cycles Elngatin 72 60** Final extensin 72 5:00 Hld 4 Infinite *61 degc fr psitive cntrl **30s fr psitive cntrl since the fragment expected is shrter.

44 Data: Lane 1 Lane 2 Ladder Pstive cntrl Lane 3 amye::gp35 ne R #2 Lane 4 amye::gp35 ne R #3 Lane 5 amye::gp35 ne R #4 Lane 6 Negative cntrl It is seen that the experiment did wrk, e.i. the cells were lysed (the psitive cntrl is as expected). A transfrmants frm #2 have the insert. Results and Cnclusin: The cpcr cnfirmed that we have ne successful transfrmant.