PALNhis: Nhe I, Xho I. PALChis: Esp3 I, Xho I. PALChisNhis: Nhe I, Xho I
PAL protein extractions from Top10. Minipreps of pBADChis, pBADNhis
Minipreps extraction of pBADChis, pBADNhis. pBAD restriction with NcoI and XhoI (Chis), NheI and XhoI (Nhis). pBAD-PAL cloning. Colony PCR
Minipreps: pBAD_PALChis 6, 7, 4 pBAD_PALNhis 2, 11 pBAD_PALNhisChis 1, 2 Miniprep extraction: 1. pBAD_PALNhisChis 1 2. pBAD_PALNhisChis 2 3. pBAD_PALChis 2 4. pBAD_PALChis 4 5. pBAD_PALChis 6 6. pBAD_PALChis 7 7. pBAD_PALNhis 2 8. pBAD_PALNhis 5 9. pBAD_PALNhis 9 10. pBAD_PALNhis 11
Analysis of sequencing results. Restriction. Transformation of pBAD_PALChis 2; pBAD_PALChisNhis 1; pBAD_PALChisNhis 2 Strains: DH10B; Top10; BL21.
Colony PCR (screening) pBAD_PALChis pBAD_PALChisNhis 1 pBAD_PALChisNhis 2
Colony PCR repeated. Selected correct colonies and prepared for protein expression assay.
Placed to grow in LB until: 1) OD = 0.1; 2) OD = 1 In 20ml
SDS-PAGE and preparation for Western blot.
Western blot. Minipreps of pETDuET NhisCstrep; pETDuet NstrepChis.
Plasmid extraction with DNA extraction kit.
Extraction of new pET plasmids. Verification with Eam1105I and NdeI.
Cloning and transformation to DH5alpha strain.
Transformation of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag in DH5alpha
Selection of corrent colonies and inoculation in minipreps for DNA extraction.
1. Miniprep extraction • Extraction of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag. Concentration 30 ng/µl; • Need to cut ̴ 10 µg * with FD NcoI and FD XhoI Concentation = 7,8 ng/µl 2. psB1C3 Picked 5 – 8 pSB1C3 and ran an agarose gel: 3. Ligation with T7 polymerase
Prepare and autoclave growth medium: 20ml x2.
Minipreps purification (RFP + pSB1C3). Bacteria are pink in liquid medium. Centrifuged 10ml (in 15ml tubes). Prepared 9 RFP (1) minipreps for tomorows purification. Antibiotics Cm. We got oligonucleotides: channels (PheP), tRNR. Made PCR with a temperature gradient for PheP, tRNR biobrick, DH10B tRNR, cProm tRNR. Polymerase: Phusion.
Minipreps purification. Yesterday’s PCR succeeded. Purification with PCR purification kit. pSB1C3 + RFP restriction. RE: EamII05I, NcoI, EcoRI, PstI. Fragments purificated through column. Transformed terminal sequence (biobrick) from kit plate 3 (3F). → DH10B Insert and vector ligation. Vector: pSB1C3 Insert: PheP, tRNA B, tRNA D, tRNA C. Transform into DH10B.
Yesterday’s transformation and PheP cloning did not succeed. Colony PCR. PheP and terminator transformation. Competent cells DH5α. pSB1C3 + RFP miniprep purification. Restriction with FD EamII05I, FD NcoI, FD EcoRI, FD PstI Fragments cutting: PheP and tRNA D with FD EcoRI and FD PstI Fragments and vectors after RE purification through column Ligation. PheP into pSB1C3, tRNA with promoter DH10B into pSB1C3 PheP, tRNA D and terminator transformation. Colony PCR did not succeed. Colonies are red that means RFP remained.
Yesterday’s transformation did not succeed. Today we are repeating experiment with other RE. Vector: pSB1C3; RE: FD EamII05I, FD PstAI, FD EcoRI, FD PstI.
Miniprep DNA extraction (pSB1C3+RFP) Digest with EcoRI and PstI Colonies PCR Arrived new genes: modified gp45 is csm4. Preparation for cloning with PCR. Phusion polymerase.
Miniprep DNA extraction: YFP, RFP, PheP, tRNAB, tRNAD, tRNAC, TERM. Digestion of pSB1C3. Ligation Transformation pSB1C3+tRNAB, pSB1C3+tRNAD, pSB1C3+tRNAC, pSB1C3+PheP. PCR of GP45 and Csm4. Purification with PCR purification kit.
Preparation of minipreps. Extraction of DNA from minipreps with TERM plasmids. Ter2 digested with FD EcoRI and FD PstI.
Prepared minipreps (pET-Duet, pET21b). Extract TERM2 from minipreps with DNA extraction kit. Digest pSB1C3. FD EcoRI with FD PstI Purification of pSB1C3. Digestion of pETDuet and pET21b with XhoI and NcoI Ligation and transformation of PolyPhe proteins.
pSB1C3-TERM-PheP, tRNAC and D colonies PCR. Cloning modified Gp45 into pET. Transformation of PheP, tRNA into pSB1C3.
Colonies PCR: Csm4 in pET and Csm4, Gp45 in pSB1C3 Extracted plasmids: pSB1C3-TER-PheP, pSB1C3-TER-tRNAD, pSB1C3-TER-tRNAC. Colonies PCR: GP45 in pET.
Extraction DNA from minipreps
Cloning Gp45 into pSB1C3, Csm4 into pET. Prepared minipreps of pSB1C3-PheP-tRNAB, pSB1C3-PheP-tRNAC, pSB1C3-PheP-tRNAD PCR of plasmids.
Transformation pSB1C3-Gp into DH5alpha.
Check Gp in pSB1C3 with colonies PCR. Digest Csm for cloning into pET. Sequencing results of Gp, PheP, tRNAC, tRNAD.
DNA extraction pET with Csm prepared for cloning. Ligation with T7 polymerase.
Transformation of PheP with tRNA. Cloning of Csm into pET. Prepared samples for sequencing.
Extraction of DNA from minipreps Csm in pET. Cloning of PheP into pSB1C3+term+tRNA. Digestion of Csm in pET, tRNA+PheP.
SDS-PAGE+NB of gp45 modified genes. Colony PCR. Run gel with restricted biobricks + colony PCR (psB1C3-T-PheP) from other not checked colonies. Also, specify PheP PCR after cleaning. Restrict csm+pETDuet, check if fragment lengths are right. If so, inoculate more cells for sequencing. Transform to strains BL21, C41, ER2566, inoculate overnight culture. Reclone pSB1C3-T-PheP if, after colony PCR, colonies will be found bad. (best to reclone right away, in case of bad results after colony PCR). (With the condition that yesterday’s cloning went wrong. There are few colonies that need to be checked by colony PCR. We have to perform colony PCR for constructs: pSB1C3-T-PheP-tRNA. There are few colonies from cloning. We have to check if constructs: Csm4 in pSB1C3 (sequencing) and gp45 in pSB1C3 (colony PCR picture) are good. Maybe we remake them. If everything is ok, check if we have enough for sequencing. If not, inoculate more for minipreping.
Yesterday’s was cloning were fine. Today, Colony PCR (pSB1C3-PheP-term-tRNA). Other colony PCR’s have been started (pSB1C3-PheP-t), 4 colonies, with the right size selected. Minipreps were inoculated. Fragment PheP PCR cleaned through column. Run (2016-09-22 colony PCR) gel again. (Gp in pSB1C3)
Preparation of samples for sequencing.
Insert tRNA to pACYC vector. We will investigate PAL + PheP performance under intestine conditions. Check GP45 perofrmance. Minipreps tRNA-T-pSB1C3. Transformation of PAL Chis. Inoculating: Cprom-term2, DH10B-term1, PheP-term 1,8 , tRNA Cprom term PheP 8, tRNA DH10B term PheP 2,4,5.
Extraction of plasmids. Digested in order to verify. Prepered fragments fragments extracted from gel. Analysis of sequencing results.
We have picked right constructs from sequencing. Cloning: PheP-term-8 + pACYC, PheP-term –tRNA DH510B 4 + pACYC, Cprom-term + pACYC, DH10B-term + pACYC. Tansforming to expression strains: PheP-term-8 to TOP10, BL21, ER2566. Growing cells for protein expression. Repeating PheP-term-8 transformation to experiment strains, because last transformation was not successful.
pBAD – PALC + pACYC-PheP was cloned. Preparations for Csm WB, pBAD-PALC + pACYC-PheP activity assay were done. Intestine contidionts experiment.