Team:Bielefeld-CeBiTec/Notebook/Selection
Lab Notebook
Documenting the Selection
Selection
Finding phase, we searched for good selection systems.
Futher literature researches for more bacterial selection methods.
Boring, but further literature researches only.
We found the bacterial two-hybrid system as the best variant for us. Literature especially for the bacterial two-hybrid system
The first thoughts were made. Finding phase for the different domains of the system.
Discussion for using RpoA or RpoZ as activation domain domain. Also the binding domain is unclear
Decision for RpoZ as activation domain and cI as the DNA binding domain Special search for the sequences.
Sequences for RpoZ and cI of phage 434 are created. Now the search for the positive controls startet.
The best hint is the new paper of Bedran et al. of 2016 which worked with a functional bacterial two-hybrid system in E. coli . Another option would be the regulator proteins Gal4 and Gal11P of Saccharomyces cerevisiae as positive controls.
Positive controls are found. HA4 and SH2 are the choices together with Gal4 and Gal11P.
The fusion proteins are designed.
E-mail contact with Ahmed Bedran for informations about their constructed two-hybrid system.
With the help of Ahmed, the Reporter was designed.
All constructs are designed and ordered by IDT as gene fragments.
Confrontation with the split-beta-lactamase and hitch-hiker system as alternatives for our selection system.
Literature researches.
Waiting for the gen synthesis of IDT.
Design of the split-lactamase and hitch-hiker with geneious.
Order of primers for fast start in the cloning processes.
More primer design and ordering.
Gen synthesis has arrived in our lab.
Design of the starting experiments. Structuring the rest of the procedure.
Lab work has started!
- Colony PCR< with Backbone pSB1C3 with primers pSB1C3_BB_fw2 and pSB1C3_BB_rv
- DpnI with PCR products.
- Gel elektrophoresis with PCR products
- Gibson Assembly with PCR products and gensynthesis orders
- Transformation in DH5α
- Sequencing of BBa_K2082201 and BBa_K2082208
- Transformation in DH5α
- First BioBricks ready
- Repeat Gibson Assembly with pSB1C3 and genesynthesis
- Transformation in DH5α
- Sequencing of parts
- Gibson assembly of dnaQ, dam, seqA, C3 backbone
- Transformation in KRX compis
- Colony PCR with the Gibson clones -> no bands
- Q5 PCR
- template pSBIC3:dnaQ, primer CH35/36, temp = 59.9 °C
- template pSBIC3:dam-seqA, primer CH37/38, temp = 58.3 °C
- template pSBIC3:dam-seqA, primer CH39/40, temp = 57 °C
- template pSBIK3:J04450, primer CH30/31, temp = 67 °C
- DPN1 digestion and gel extraction of pSBIK3 backbone
- Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)
- Transformation in DH5α
- Colony PCR of ASI with VR/VF -> plating positive clones out
- Sequencing of positive clones
- Q5 PCR
- template pSBIC3:emrR, primer BF1/2
- template pSBIC3:ugi-cda1, primer BF4/5
- template pSBIC3:emrR, primer BF6/7
- template pHSG:EPPolI, primer CH42/43
- template pHSG:WTPolI, primer CH42/43
- PCR clean up of the Q5 PCR
- Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)
- Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone
- Transformation in DH5α
- Transformation of pSBIC3:K584001 & K608010
- Planning of reversion experiments
- Sequencing results: mutagenesis ASI has mutations, ASII did not work
- Transformation of ASII Gibson
- Plasmid isolation of ASI/ ASII clones
- Colony PCR of ASII
- Restriction digestion of positive clones of ASII
- Repeated colony PCR of ASI with primer cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR
- Restriction digestion of right ASII clones with EcoR I/Pst I
- Restriction digestion of right ASI clone with EcoR I/Pst I
- Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)
- PCR for coloning ASI into pSBIC3
- template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6 °C
- template pSBIC3:RFP-cd, primer CH30/31, temp = 67 °C
- template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6 °C
- DPN1 digestion
- Restriction digestion with EcoR I/Pst I
- pSBIK3:J04450
- pSBIC3:K608010
- pSBIC3:K584001
- Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
- Restriction digestion with EcoR I/Pst I of ASII and pSBIC3:Strong RFP
- Ligation of ASII and strong RFP
- Transformation of the ligation into DH5α
- Gel extraction of pSBIK3, K608010, K584001
- Ligation of pSBIK3 + K608010
- Overnight culture of the ligation
- Gel extraction of PCR products & PCR clean up
- Gibson assembly
- pSBIC3:AraC-Pbad (non-leaky)
- C3 backbone + ASI clone 4
- C3 backbone + ASI clone 15
- Ligation & Transformation in DH5α
- Colony PCR
- template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58 °C
- template pSBIC3:ASI, primer VR/VF_rev, temp = 58 °C
- Q5 PCR
- template A68T_A, primer CH20/A68T_rev, temp = 58.6 °C (AA)
- template A68T_B, primer A68T_B/CH21, temp = 58.5 °C (AB)
- template C379A_A, primer CH20/C379A_rev, temp = 57.7 °C (BA)
- template C379A_B, primer C379A_fw/CH21, temp = 58 °C (BB)
- template A380T_A, primer CH20/ A380T_rev, temp = 58.1 °C (CA)
- template A380T_B, primer A380T_fw/CH21, temp = 58.1 °C (CB)
- Gel extraction
- Gibson assembly
- Transformation in KRX
- Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64
- Colony PCR of AraC-Pbad (non leaky), ASI
- Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C
- Sequencing of pSBIC3:emrR-1/-2 with VR/VF
- Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50 °C
- Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010
- Restriction digestion of C3 backbone with EcoR I/Pst I
- Transformation of pSBIC3:K516031 in KRX
- Colony PCR of pSBIC3:K608010_StopA/B/C with VR/VF
- Sequencing A with VF, B with VR and C with VR
- Plasmid isolation of Stop-GFPs -> Sequencing
- Restriction digestion
- pSBIC3:AraCPbad with Spe I, Pst I
- pSBIC3:AraCPbad(non-leaky) with Spe I, Pst I
- pSBIC3:RFP Generator with Xba I, Pst I
- pSBIK3:MP6-I-Klon 4 with Spe I, Pst I
- pSBIK3:MP6-II-Klon with Xba I, Pst I
- Designing of new primer
- Gel extraction of the restriction digestion
- Ligation
- C3:AraC-Pbad + RFP-Generator
- C3:AraC-Pbad(non-leaky) + RFP-Generator
- K3:Mut-I + Mut II
- C3 + MutII
- Repeat of restriction digestion of ASII: template K3:ASII, EcoR I-Hf/Pst I
- Gel extraction of the restriction digestion
- Ligation
- Transformation of the ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
- Colony PCR with VR/VR of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII
- Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose
- Q5 PCR , temp = 57 °C
- template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61
- template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57
- template C3:dnaQ, primer CH54/CH55
- template pHSG-WTPolI, primer CH58/C59
- template pHSG-WTPolI, primer CH42/C43
- template pHSG-EPPolI, primer CH58/C59
- template pHSG-EPPolI, primer CH42/C43
- DPN1 digestion
- Gel extraction
- Gibson assembly
- damseqA + C3
- ugi-cda1 + C3
- WTPolI-Part + C3
- EPPolI-Part + C3
- dnaQ-Insert + dnaQ-BB
- WTPolI-Expression + PolI-BB
- EPPolI-Expression + PolI-BB
- Transformation
- Colony PCR with VR/VF, temp = 57 °C
- C3:AraC-Pbad(tight)-B0031-WTPolI
- C3:ugi-cda1
- C3:AraC-Pbad(tight)-B0031-dnaQ
- C3:EPPolI
- C3:AraC-Pbad(tight)-B0031-EPPolI
- C3:seqA
- C3:WTPolI
- Sequencing
- Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10
- Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10
- Colony PCR of ugi-cda1 -> Sequencing of right clones
- Q5 PCR , temp = 58 °C
- template C3:damseqA, primer CH71/72
- template C3:StoppGFP Stop A Stop B, primer CH20/66
- template C3:damseqA, primer CH73/74
- template K3:Stopp GFP A+B, primer CH64/21
- DPN1 digestion of the Q5 PCR
- Gel extraction of Stop A, Stop B
- Gibson assembly stop A + stop B
- Restriction digestion with EcoR I/Pst I ASI+ASII, GFP_Astop_2, GFP_Astop_2
- Transformation of C3:PRha, C3:PRha-GFP-Generator in KRX
- Transformation of PRha-GFP-Generator in top10
- Transformation of StoppA+B in DH5α
- Q5 PCR , temp = 58°C
- template C3:dnaQ, primer 54/55
- template C3:WTPolI (CWP1), primer 58/59
- template C3:EPPolI (CEP1), primer 58/59
- template C3:ASI+ASII, primer 54/76
- template C3:K516031, primer 56/57
- template C3:K516031, primer 60/61
- template C3:K516031, primer 60/61
- template C3:K516031, primer 75/57
- DPN1 digestion
- Gel extraction
- Gibson assembly of seqA
- Transformation in DH5α of C3:seqA, K3:StopA+B
- Q5 PCR of C3:damseqA with CH71/72, temp = 58 °C
- DPN1 digestion
- Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB
- Transformation of the Gibson assembly in DH5α
- Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
- Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
- Colony PCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR
- Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter
- Q5 PCR of C3:damseqA, primer 71/72, gradient 55-59 °C/li>
- Sequencing of stop A+B
- Restriction digestion of K3:ASII, C3:Terminator with EcoR I, Pst I
- Gibson assembly of M6 in RFPGen -> Transformation
- Test cultivation of a 96-well plate
- Colony PCR of M6-Gen -> Plasmid isolation
- Restriction digestion of dnaQ-Gen with Xba I/Pst I
- araE-PCR with temp 58 °C
- Gibson assembly of araE
- PCR of DNA, primer 81/82 and C3:med RFP 83/84
- Gel extraction of dnaQ-Gen
- Ligation with C3:AraC-Pbad/C3:PRha
- DPN1 digestion
- PCR clean up
- Gibson assembly of araE-Insert + araE-backbone
- Transformation of the ligation in KRX
- Restriction digestion of C3:M6-Gen with EcoR I/Xba I, weak/RFP Gen with Xba I/Pst I
- Colony PCR of C3:araE-Device
- Gibson assembly of C3 backbone + ugi_cda1, C3 backbone + ASII
- Transformation of the Gibson assembly in DH5α
- Colony PCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF
- Restriction digestion of M6-Generator with Xba I/Pst I, C3:K808000, C3:Prha with Spe I/Pst I
- Clean up
- Ligation with backbone
- Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI
- Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37 °C
- Transformation of C3:AraC(tight)dnaQ + plA230 in Top10
- Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ
- Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ
- Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2 h growth, +25mM arabinose/25mM glycose
- Transformation of JS200 EP/WT with plA230
- Colony PCR of AraC-Pbad-M6; Prha-M6
- Transformation of plA230 in JS00 EP/WT
- Rifampicillin assay
- beta-lactamase Reversions assay
- Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10
- Overnight cultures
- Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10
- Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture
- Isolation of JS200 + PolI+plA230
- Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)
- Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation
- Restriction digestion of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6
- Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)
- Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
- Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
- Reversions assay
- Restriction digestion of E/P of pSB4C5(E/P)
- Q5 PCR
- template C3:ugi-cda, primer VF/68, temp = 58 °C
- template C3:cda, primer 69/70, temp = 60 °C
- template A3, primer CK06/05, temp = 59.9 °C
- template reporter, primer CK07/08, temp = 59 °C
- Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10
- Reversions assay
- Analysis reversion assays
- Analysis of MiSeq data
