Team:Manchester-Graz/Notebook
Notebook
Week 1 (29.6.2015 - 5.7.2015)
- Selection plates (kanamycin) for AADC and CvATA
- Transformation of BL21 with AADC and CvATA
- Colonies grown on selection plates
- Left in the fridge over the weekend for starter culture to be obtained on Monday
Week 2 (6.7.2015 - 12.7.2015)
- Starter culture for AADC and CvATA
- Expression culture for AADC and CvATA
- Induction with IPTG
Figure 1 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. EsaRv2 = pJET_EsaRv2 cut with
HindIII/SacI, expected insert size 863 bp; cepRv2 = pJET_cepRv2 cut with EcoRI/EcoRV, expected
insert size 944 bp; EsaR = pJET_EsaR cut EcoRI/EcoRV, expected insert size 960 bp. The marked
samples (green arrows) were sent for sequencing. Std: NEB 2-Log DNA Ladder
Figure 2 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. EsaI = pJET_EsaI cut with
BamHI/PstI, expected insert size 765 bp; CFP = pJET_CFP cut with BamHI/HindIII, expected insert
size 934 bp; RFP = pJET_RFP cut SacI/BamHI, expected insert size 1104 bp. EsaRv2 = pJET_EsaRv2
cut with HindIII/SacI, expected insert size 863 bp The marked samples (green arrows) were sent for
sequencing. Std: NEB 2-Log DNA Ladder
Figure 3 Different gBlocks were cloned into pJET and transformed into E. coli TOP10. To verify correct
cloning the plasmids were digested to analyse proper insert size. bla = pJET_bla cut with EcoRV/SpeI,
expected insert size 1083 bp; cepR = pJET_cepR cut with SacI/HindIII, expected insert size 843 bp;
EsaI = pJET_EsaI cut BamHI/PstI, expected insert size 765 bp. The marked samples (green arrows)
were sent for sequencing. Std: NEB 2-Log DNA Ladder
- This week we blunt end ligated eight of our gBlocks (esaR, esaR v2, cepR, cepR v2, esaI, CFP, mRFP, bla) that are needed for the assmebly of two different variants of pCERI into pJET 2.1 respectively, transformed them into E. coli TOP 10 and plated some of the obtained transfomants on LB - Amp for plasmid preps.
- Those plasmid preps were cut with the corresponding restriction enzymes and an analyzing gel (Fig. 1-3) was run to check for correct size of the inserts.
- Correct plasmid constructs (marked with a green arrow) were sent for sequencing.
- The remaining plasmid DNA of the 11 correct vectors was cut and purified to be used for future OE-PCR
Week 3 (13.7.2015 - 19.7.2015)
- Whole cell biotransformation of AADC and CvATA
- test
- test
- Sequencing results of week 1 were evaluated. For all gBlocks, except for esaRv2 correct constructs were found.
- Plasmid preparations of the correct gBlocks were digested and inserts of the correct size were cut out of the analyzing gel and purified.
- New ligations of esaR and esaRv2 with pJET 1.2 were performed and transformed into E.coli TOP 10. Transformants were streaked out again. After plasmid isolation they were again digested to check for the correct size of the inserts. Plasmid preparations containing the correct size were sent for sequencing. All of the obtained sequences showed mutations or deletions (Fig. 1).
Figure 1 Part of a sequencing result of EsaRv2. The sequence contains a deletion at position 618. As the mutation is in the coding region of the
gene the corresponding sequence could not be used for further work.
- Thus the experiment was repeated.
- pPIC9 was digested with BglII to cut out the E.coli origin of replication as well as the Amp-resistance marker. The linearized vector was further dephosphorylated and purified.
- OE-PCR of bla_p15A was performed. As the first attempts resulted in some unspecific bands on the analyzing gel (Fig. 2), the PCR was repeated with more restrictive conditions. Even though an unspecific band remained, a band of the correct size (~2 kb) was obtained and purified. The fragment was digested with BglII to allow cloning into pPIC9.
Figure 2 Overlap extension PCR of bla_p15A resulting in unspecific bands;
Std: NEB 2-Log DNA Ladder
- bla_p15A was cloned into pPIC9. The resulting circular vector was transformed into E.coli TOP 10 and plated on LB-Amp. However no transformants were obtained, putatively due to the very low concentration of the ligation product. Transformation was repeated with a higher amount of the ligation.
Week 4 (20.7.2015 - 26.7.2015)
- bla bla
- bla bla
- After sequencing multiple clones of pJET_esaR and pJET_esaRv2 that all contained different mutations we decided to take one clone of each construct that only contained a single deletion, go on with the experiment and cut out the sequenced gBlocks with the corresponding restriction enzymes.
- OE-PCR with the digested and purified fragments of esaR, cepR and esaI as well as with esaRv2, cepRv2 and esaI were performed to obtain the two fragments esaR_cepR_esaI and cepR_EsaR_EsaI_v2, that basically contain the same genetic information, however in a shuffled order. The fused gene fragments was afterwards blunt-end cloned into pJET 1.2. Correct cloning was verified by a restriction digest and an analyzing gel (Fig. 1).
- As both esaR_cepR_esaI as well as cepR_esaR_esaI_v2 still contained esaR-sequences with deletions site, directed mutagenesis with a primer-pair for each construct was performed to correct the sequence at the respective positions. After the PCR the methylated template plasmids were digested with DpnI to minimize the background still containing the deletions. After digestions, the corrected vectors were transformed into E. coli Top10.
- Last weeks OE-PCR of bla_p15A was repeated to obtain more of the fragment. The PCR product was purified and digested with BglII. The gene fragment with the sticky ends was ligated again into pPIC9 that had been prepared last week. After ligation the circular vector was transformed into E. coli Top10.
- This week pPIC9_p15A_bla transformation resulted in way more transformants. Plasmid isolation of several constructs was performed. To check for the correct insert the vector was digested with SacI and BamHI (Fig.1) . Correct vectors resulted in ~5600 bp and a ~2000 bp band. Clone 9 was found and used for further experiments.
Figure 1 Analyzing gel of digested vector constructs; Overlap-extension PCR products were blunt-end cloned into pJET 1.2 and transformed into E.coli Top 10. To verify correct cloning, the plasmids were digested to analyse proper size. Std.: Quick-Load Purple 2-Log DNA Ladder; v1: pJET_EsaR_CepR_EsaI_v1 cut EcoRI/SacI, expected insert size ~ 1800 bp; v2: pJET_CepR_EsaR_EsaI_v2 cut EcoRI/SacI, expected insert size ~ 1800 bp; pPIC9: pPIC9_p15A_bla cut PstI, expected insert size ~ 2000bp; 1-11: plasmid preps 1-11of different vector constructs; X: empty slot
Week 5 (27.7.2015 - 2.8.2015)
- bla bla
- bla bla
- Due to some delay in the gene fragment synthesis, finally our last gBlock cepI arrived. We cloned it into the pJET vector with blunt-end ligation. It was then transformed into E. coli TOP10 for plasmid amplification.
- The two resulting clones (M1 and M2) were plated for plasmid preparations. The obtained DNA was cut for an analyzing gel using PstI and NotI. The bands fit the expected size of around 900 bp (Fig. 1) and were sent for sequencing.
- The sequencing results showed that one of the clones (M1) contained no mutations.
Figure 1 pJET_CepI (M1 and M2) cut with
PstI and NotI on the control gel.
- The remaining plasmid preparation of M1 was cut with NotI and SpeI for overlap extension PCR (OE-PCR) and purified on a preparative gel.
- That cepI was then used for OE-PCR with cfp. The PCR product was run on a gel. It showed the expected bands at 2000 bp. The DNA was extracted from the gel slices and ligated into pJET. The resulting plasmid was used for transformation into E. coli TOP10.
- This week we set up our controls as well. We transformed pSB3C5_BBa_J04450 (mRFP) and pSB1C3_BBa_J04421 (cfp) from the distribution kit 2015 into E.coli Top10 and isolated the plasmids. To get the same vector background with the same origin of replication (p15A) as our pCERI-vector, we cut both constructs with EcoRI/PstI. After digestion pSB3C5 was also dephosphorylated. BBa_J04421 was cloned into pSB3C5 afterwards.
- The size verified pPIC9_p15A_bla clone was streaked out again to isolate more plasmid. After plasmid isolation the vector was cut with EcoRV and XbaI to get the p15A_bla fragment for the Gibson assembly of pCERI.
Week 6 (3.8.2015 - 9.8.2015)
- bla bla
- bla bla
- bla bla
Figure 1 Analyzing gel of pJET_cfp_cepI cut PstI/SacI. Correct plasmids should yield bands at 2669 bp,
1733 bp and 416 bp.Std: NEB 2-Log DNA Ladder; 14-17: plasmid preps of different clones;
- Plasmid isolation of pJET_cfp_cepI was performed and correct cloning was verified by restriction digestion with SacI and PstI.
- On the analyzing gel (Fig. 1) we saw the three bands we expected: One just below 3000 bp (vector backbone), one around 1700 bp (cfp_cepI) and one at 400 bp (smaller part of the backbone).
- The clones 14 and 17 were sent for sequencing. Clone 17 showed no mutation. The remaining plasmid of clone 17 was cut with HindIII and SpeI for a preparative agarose gel. The resulting band for cfp_cepI was purified to be further used for the Gibson assembly of pCERI.
- We conducted plasmid isolation of transformants containing pSB3C5_BBa J04421 (cfp) and size verified correct cloning by restriction digestion. Correct clones are stored on 4°C to be used as positive controls for the analysis of pCERI.
- As the previous site directed mutagenesis attempts to correct the mutation in esaR did not get us the results we hoped for and only showed the same deletion as the template DNA we repeated the site directed mutagenesis-experiment with altered experimental parameters, prolonging the DpnI digest from 1h to 12h (overnight) to make sure to get rid of all template DNA of the PCR. This resulted in significantly less clones on the LB-Amp plates after transformation into E.coli Top 10. These clones will get sequenced next week.
- This week we also made our first Biobricks. PCRs to attach the Biobrick suffix and prefix to the sequence verified gBlocks of PaidA_mRFP, PesaRC_cfp, cepI, esaI and cepR were performed. PCR-products were digested with EcoRI and PstI and purified. All products were ligated into linearized and dephosphorylated shipping vector pSB1C3. pSB1C3_BBa_K1670004 showed no transformants on the plates and thus transformation was repeated. Clones of all other constructs were streaked out again for plasmid preps and correct cloning was verified by restriction digests (Fig. 2-4). Clones containing size verified inserts were sent for sequencing.
Figure 2 pSB1C3_BBa_K1670003(encoding mRFP)cut EcoRI/PstI;
Std: NEB 2-Log DNA Ladder; 1-4: plasmid preps of different clones;
Figure 3 pSB1C3_BBa_K1670000 (encoding cepI) cut
EcoRI/PstI; Std: NEB 2-Log DNA Ladder; 5-7: plasmid preps of different clones
Figure 4 pSB1C3_BBa_K1670001 (encoding cfp) and pSB1C3_BBa_K1670002 (encoding cepR) cut EcoRI/PstI; Std: NEB 2-Log
DNA Ladder; 5-7: plasmid preps of different clones
Week 7 (10.8.2015 - 16.8.2015)
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
Figure 1 pSB1C3_BBa_K1670004 (encoding esaI)
cut EcoRI/PstI with an expected insert size of ~700 bp;
Std: NEB 2-Log DNA Ladder; 1-2: plasmid preps of
different clones
- Sequencing reports for the size verified biobrick parts of last week showed no mutations in all sent samples, meaning we have already four biobricks ready. Transformation of pSB1C3_BBa_K1670004 was repeated. Obtained transformants were streaked out again and their plasmids isolated. Correct cloning was verified by restriction digests with EcoRI /PstI and run on an analyzing gel (Fig.1). Sequencing of both clones showed no mutations in clone 1, verifying our fifth biobrick.
- Sequencing results of the repeated attempt of site directed mutagenesis of esaR showed that we were able to correct the deletion. Additionally, we also sequenced the other genes of the fragment esaR_cepR_esaI that also verified the sequence of the OE-PCR. Thus this fragment was excised from the pJET backbone and purified.
- Furthermore, the sequence verified pJET_esaR_cepR_esaI clone was used as a template for PCR to attach the biobrick pre- and suffix to esaR to generate our last biobrick pSB1C3_BBa_K1670005.
- After the successful site directed mutagenesis experiment all fragments for the Gibson assembly of pCERI (PaidA_mRFP, PesaRC_cfp_cepI, PesaS_esaR_cepR_esaI and bla_p15A) were finally available. Gibson assembly was performed according to the protocol: 0.05 nmol of each fragment were used for the reaction. 2µl of the reaction mix were transformed into E.coli TOP10. Plated transformants were grown over the weekend.
- Additionally this week we also started to set up the vectors needed for the characterization of our biobricks. BBa_K1670001and BBa_K1670003 each were cloned into pSB3C5 to receive a vector backbone with a lower copy number of 10-15. Further J61002_J23100 from the distribution kit was transformed into E.coli TOP 10. After plasmid isolation the vector was cut with SpeI and PstI and dephosphorylated to be able to clone the regulatory genes of our quorum sensing systems BBa_K1670005 and BBa_K1670002 right after the constitutive promoter BBa_J23100 next week.
- New electrocompetent cells of E.coli BL21 and E.coli Nissle 1917 were made according to the same protocol used for E.coli Top 10 and a test transformation with both strains was performed. Transformation efficiencies of 2,3*108 cfu/ml for E.coli BL21 and 3,7*108cfu/ml for E.coli Nissle were reached.
Week 8 (17.8.2015 - 23.8.2015)
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
Figure 1Analyzing gel of putatively assembled pCERI
plasmid prep cut with multiple restriction enzyme
combinations. 1: pCERI cut BamHI with an expected
bands at 3579 bp, 1989 bp and 1617 bp; 2: pCERI cut
EcoRI/PstI with expected bands at 3493 bpm 2499 bp
and 1193 bp; 3: pCERI cut PstI/HindIII with expected
bands at 2102 bp, 1790 bp, 1703 bp and 1590 bp
Std.: NEB 2-Log DNA Ladder
- BBa_K1670005 and BBa_K1670003 were ligated into J61002_J23100 and transformed into E.coli Top 10. Transformants were streaked again for plasmid isolation. Correct cloning was verified by digestions with EcoRI and PstI. Correct plasmids were cotransformed into E.coli BL21 and single colonies were streaked on LB-Amp.
- Plasmid isolation was performed with colonies from last week’s Gibson assembly. The gained plasmids were cut in different combinations (Fig.1) to check for the correct assembly of pCERI. One clone showed correct bands for all different approaches and was sent for sequencing.
- The putatively correct pCERI was transformed into E.coli BL21 and E.coli Nissle 1917 and single colonies were streaked on LB-Amp.
- pSB3C5_J04421 and pSB3C5_J04450 were also transformed into E.coli BL21 to be used as positive controls later on.
Week 9 (24.8.2015 - 30.8.2015)
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
- bla bla
- This week we started characterization of pCERI. To proof expression of the regulatory proteins as well as our genes of interest, fermentations were conducted. Overnight cultures were grown with single cell colonies of E.coli BL21 pCERI (due to a limited amount of space in our incubator, fermentation is only performed with E.coli BL21 pCERI, while E.coli Nissle 1917 will be cultivated next week). 300 ml of LB – Amp were inoculated to a starting OD600 of 0.05 and incubated at 37°C and 100 rpm. As positive controls recombinant E.coli BL 21 pSB3C5_J04421 and pSB3C5_J04450 are cultivated under the same conditions. As negative controls wildtyp E.coli BL21 as well as a sterile control containing only LB-Amp without any inoculum, are used. Cultivation was stopped after 1, 2, 3, 4, 5 and 6 hours and cultures stored on ice till the cells were harvested by centrifugation at 4000 rpm for 10 minutes.
- Table 1 OD600 after harvesting of the cells after different incubationCell disruption was performed by sonication. An insoluble protein fraction was separated by centrifugation. Protein concentration of each sample was estimated by Nanodrop spectrometry (Tab. 1). Proteins of each sample, normalized to an OD600 of 0.7 were separated by SDS-poly-acrylamid gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue(Fig. 1). As the negative control ( E.coli BL 21 (-))showed very poor growth during fermentation, this sample is still missing on the gel and thus the SDS-PAGE should be repeated next week. Furthermore, three SDS-gels of other research groups were conducted simultaneously to ours and resulted in a poor quality. Thus, the SDS-PAGE should be repeated anyways. However, we already see that our CFP positive control ( E.coli BL 21 pSB3C5_J04421 ) seems to show no expression of CFP at all, as no band can be seen at 26.8 kDa as opposed to the strong band of mRFP at 25.4 kDa. This confirms an old user review of BBa_J04421 ) , that this part might not work properly.
intervals and protein concentration, measured with Nanodrop at
280 nm, after cell disruption; PC_mRFP: positive control mRFP;
PC_CFP: positive control CFP
Figure 1 Stained SDS-poly-acrylamid-gel of cell lysat samples of E.coli BL21 pCERI
after different incubation intervals (1h – 6h) all normalized to an OD600 of 0.7; CFP:
E.coli BL 21 pSB3C5_J04421 induced with 0.1 mM IPTG, however the part seems
to show no expression of CFP; mRFP: E.coli BL 21 pSB3C5_J04450 induced with
0.1 mM IPTG - This week also the first try of the fluorescence assay for the characterization biobrick parts BBa_K1670002 (cepR) along with BBa_K1670003 (PaidA_mRFP) and BBa_K1670005 (esaR) along with BBa_K1670001 (PesaRC_cfp) was performed according to our protocol with HSL concentrations ranging from 0.01 mM to 100 nM. Already at very low C8-HSL concentrations all samples containing BBa_K1670003 (PaidA_mRFP) and at low concentrations of 3OC6-HSL samples containing BBa_K1670001 (PesaRC_cfp) showed high fluorescence emission. That might indicate, that the added HSL were diluted in wrong way and thus the experiment will be repeated next week. Once again E.coli BL 21 pSB3C5_J04421 induced with 0.1 mM IPTG showed hardly any fluorescence at 476 nm confirming the result of the SDS-PAGE.
