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XylE team Lab Objectives
- construction of XylE fusion protein
- Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter
- Construction of the -ComE promoter/XylE fussion protein- expression system
- Construction of the -LacI promoter/XylE fusion construct- expression system
Lab notes and schedule
- anealing DNA strands of J23101 promoter in a waterbath
we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assemply to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted biobricks.
- prepared two overnight cultures of XylE transormed E.coli (one 50microliters and one of 450 microliters)
these cultures are going to be used tomorrow for mini-prepping. Miniprep will allow us to isolate E.coli's plasmid DNA(which contains the XylE gene).
- mini-prep of XylE transformed E.coli
Mini-prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasnid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini-prep will later be used for the midi-prep (that gives out higher yeilds of DNA needed for cloning).
- gel analysis of plasmid DNA retreived from mini-prep of XylE transformed E.coli, cut with restriction enzymes. From light to the left, 50micrograms digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut biobrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.
- midi-preped the XylE-transformed E.coli. The DNA yeild from the midi-prep was 134micrograms as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.
- restriction digestion of midi-prepped XylE by Xbal and PstI to prepare it for 3A assemply. (with J23101 promoter and PSB1C3 vector)
- gel analysis of the restriction digestion mixture to isolate XylE gene
- I made an overnight culture of Bacillus
- using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel lamp.
- gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine their ratios for 3A assemply ligation
- 3A assemply of XylE, J23101 promoter and pSB1C3 vector.
- Transformation of XL-Blue competent E.coli with the above construct.
*I followed Chris's Bacillus transformation protocol to transform Bacillus with constitutive GFP and RFP DNA as well as a control without DNA
- We run the XylEGFP1 PCR reaction to construct the His-GFP-Flag and Linker-XylE-Spe construct.
- we run a gel on XylEGFP1 PCR reaction. Results: GFP was extended successfully, XylE extension FAILED (too much non-specific annealing)
- catechol assay on 2hrs bench ligation of promoter,xylE and vector failed.
- two replica plates of overnight ligated J23101, XylE and pSB1C3 transformed E.coli (one for catechol assay)
- the His-GFP-Flag DNA was gel purified
- transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-xylE ligation, since we believe that the strand annealed promoter was of bad quality
- set up overnight cultures for midi-prep
- gel separation of linker-XylE-Spe DNA FAILED
- PCR reaction for extension of His-GFP-Flag
- Catechol assay on E.coli transformed with overnight ligated J23101, XylE and pSB1C3. FAILED
- midi-prepped registry obtained J23101. A yield of 130ng/ul of promoter was obtained. The promoter is in a biobrick vector called J62001. The promoter is upstream of RFP gene.
- the vector carrying the promoter was digested with SpeI and PstI, while XylE gene was digested with XbaI and PstI.
- the promoter and the XylE gene were gel purified.
- a reaction between the promoter(still on vector) and XylE was set on for overnight ligation
- PCR purification of GFP2 -> GFP construct ready for full fusion protein.
- Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62C and only rev primer to create pool of successful extensions with with rev primer (60-62). PCR round 2 with Fwd primer and temperature scale (68-68, 72-74).
Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appauling(explanation for Nick:really bad) results, 2nd gel run was successful.
- Performed a ligation reaction between the vector containing J23101, and XylE(one on bench and one overnight one).
- Transformation of the new plasmid into competent E.coli. Successfully transformed colonies can be selected for by loss of RFP expression.
- XylE-1 PCR with temperature cascade. Gel analysis and purification.
- white colonies from the promoter-XylE transformed E.coli were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate.
- XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards.
- Catechol assay performed on promoter-XylE transformed E.coli. SUCCESSFULL
- XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein.
- Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing).
- Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstins advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV).
- Mini prep of 4 x J23101-XylE taken from 4 different colonies (8 14 24 27).
- The gel analysis showed successful vector uptake.
- Set up an overnight culture for midi using colony 24 (gel analysis showed similar results colony 24 was picked randomly.)
- A new primer was designed in order to add a corrected TEV-protease-cleavable sequence to the His-GFP-Flag construct. This was controlled and ordered.
- Midi prep of colony 24 for XylE-J23101 the final concentration was ~100ng/ul which wasnt so great but Chris says the protocol produces very poor yields.
- We are performing the next building step of our vector. PSB1C3 containing terminator B0014 was cut with EcoRI and XbaI. The insert was cut with EcoRI and SpeI and both were incubated for 1.5hrs. Wolf is now running a gel to purify out the insert via gel purification and perform a PCR purification on the vector.
- Advisors have decided it's best not to use Jeremy's tagged XylE due to the 93% difference. Kirsten will be tagging the registry XylE and we shall purify and assay with that instead.
- We shall see purification expert Kieran tomorrow and talk through the process. Chris will also talk us through our characterization of XylE experiments- we will use the robot after it's been programmed but until then we can use the plate-reader.
- Spectrophotometry experiments with XylE transformed E.coli in LB medium (M9 culture was contaminated) reveiled the followings: On catechol assay of the trasformed cells, the positive yellow output can be quantitively measured by a broad peak at 380nm.
- Trasformation of competent E.coli cells with promoter-XylE-terminator pSB1C3vector.
- experiment to determine concentrations of catechol and cell density for assays
- The new GFP + TEV primer arrived, was diluted and used to set up the appropriate PCR.
- PCR extension of Pveg promoter: EcoRI---Pveg---RBS-SpeI
- I performed a catechol assay on the picked transformed colonies to deduce which ones were successfully transformed with the insert plus vector. 1-5 7 and 10 failed to turn yellow (1-5 were background controls)leaving 8 yellow colonies.
- I had to perform a colony PCR on two selected colonies 8 and 14 to check the correct insert+terminator was present.
- Colony 8 when run on a gel analysis showed the correct size 924 XylE + 97 J23101 + 35 B0014.
- This was set up as an overnight culture.
- Gel analysis of the GFP-TEV construct showed satisfactory bands.
Tuesday 7th Sept
- Mini prep of the overnight Colony 8 culture (by Wolf)
- Midi prep of overnight culture
- gel purification of PCR product EcoRI--Pveg--RBS--SpeI
- overnight restriction digestion of EcoRI--Pveg--RBS--SpeI with SpeI
- started data analysis of plate assay
- Annealing PCR reaction included 2 samples without and one with additional primers (XbaI-His-GFP Fwd, SpeI-XylE Rev). While the primer-including reaction did not show any clearly identifiable bands, the others showed clear, if very weak bands at 800 and 1000bp, which represent the GFP and XylE constructs respectively. No band was identifiable at the 1.7 kb range, which would have indicated a successful annealing reaction. However, problems with the lense of the gel-analyser were only discovered later to have severely reduced the band brightness. Potentially a PCR-reaction with appropriate primers to amplify an annealed product(XbaI-His-GFP Fwd, SpeI-XylE Rev), could have been successful. However the reaction mixture was disposed of before this became clear.
Wednesday, 8th Sep
- A second PCR extension of Pveg promoter to introduce the RBS and cut sites. It was also gel purified and stored in gel lumbs in the freezer. (maybe needed later so keep in mind).
- Overnight restriction digestion completed. Then, it was run on the gel to check if it worked and then gel purified again.
- Vector PSBI-C3 was digested to remove the terminator and make it sticky for the insert (Pveg-RBS). Then it was run on the gel to check if restriction has worked, but the gel didn't run far enough to determine easily between undigested and digested vector.
- For further annealing reactions for GFP-XylE constructs additional XylE(2) template was required -> amplification PCR for XylE(2).
Thursday, 9th Sep
- Gel with digested and undigested vector PSBI-C3 was run and then the digested vector was purified.
- Gel was run to determine the DNA concentration ratio for the ligation of PSBI-C3 and Pveg-RBS.
- Vector PSBI-C3 was dephosphorylated.
- Ligation of PSBI-C3 and Pveg-RBS has been set up overnight.
- Gel-analysis and gel-purification of the XylE(2) amplification PCR product.
Thursday, 10th Sep
- The transformation of E.Coli with Pveg-RBS in PSBI-C3 and PSBI-C3 by itself (to check see how successful dephosphoryaltion of PSBI-C3 was and estimate the percentage of bacteria that contain the insert) was completed
- concentration of the midi prep of J23101-XylE-B0014 was determined to be ~600ng/ul (using new protocol)
- Kyasha kindly digested my midi with EcoRI and SpeI and performed a gel analysis. The results show a potential additional plasmid contaminating my midi however the concentration of DNA was extremely high. NB Chris said that it could be sheared DNA from a midi prep step.
- the midi prepped plasmid was transformed into testing E.coli strain TOP10.
Friday, 10 sep
The transformation was a SUCCESS. 2x replica plates were made plate 1# 1-6 plate #2 6-11; colony 6 and colony 9 of plates 1 and 2 respectively were transfered into a 5ml liquid culture + 5ul CmR. These will later be turned into glycerol stocks. After the replica plates have grown up mini preps on a number of colonies shall be performed - this hopefully will eliminate the contaminating plasmid DNA. This will be followed by a midi prep.
Saturday, 11 Sep
The transofrmation of E.Coli with PSB1-C3 with insert did not work :(
Monday 13th september
Overnights weren't set up on sunday so they were made up alongside some assay cultures. J23101-XylE-B0014 colonies 8 and 10 of the replica plate #2 were picked. Chris also provided a replica plate containing 3k3 vector colonies. This was over a year old and he was unsure whether it was the correct plasmid or if the cells would grow up. I picked all available colonies 102 150 151 and 260 of kanamycin resistance. Lastly for assays 2x LB 2xM9 cultures were made 5ml +5ul antibiotic.
Tues 14th September
- assay on plate reader of:
- 0-0.75 initial O.D. of transformed E.coli
- 0-1mM initial catechol concentration
The assay was carried out with E.coli, top ten spcies, transformed with J23101-XylE-B0014 in pSB1C3 vector.The overnight culture wastransfered in new medium this morning for 4 hrs before assaying. LB medium was used for dilutions and blank. Catechol was diluted in ddH2O. Image:Catechol assay test 2.xls
- Mini preps of XylE 8,10 colonies and 3K3;102 151 260 colonies. Analytical digests were performed using E+S on XylE, E+S AccI and HindIII+E for 3K3 vector respectively. The band patterns correctly identified the vector to be 3k3. Colony 10 of XylE and Colony 260 of the 3k3 vector were set up direct into 100ml LB and so will require til 12pm for midi prepping on weds. Nick performed a second plate reader assay to determine the minimal concentration of Catechol to use for assays. I took 990ul M9 culture containing colony 10 and 10ul catechol was added. This was incubated for 10 mins and then spun down. The supernatant was removed into a cuvette and the cells resuspended in M9 salts. The ODs were then read on the spectrophotometer at 380 and 600nm. It was found that control cuvette of M9 salts had a miniscule reading. M9 + cells ~0.007. Supernatant + cells were ~ 2.5 therefore we could deduce that the coloured catechol breakdown product is exported out of the cells.
- Mini prep of 1 sample (6 samples lost due to mistake) of GFP-Xyle fusion protein and its digestion with Spe and Xba (gel to be run on the next day)
- Preparing E.Coli colonies for the next day for mini prep to be redone
- PCR of 6 samples of GFP-Xyle fusion
Weds 15th September
- Midi preps of overnights.
- In preparation for assays determining the effect of catechol and/or breakdown product on cell viability we prepared Top10 cells, one strain containing pVeg-XylE-terminator and the other containing a CMR plasmid of similar size. As the XylE cells have already been prepared, only Top10 transformation with CMR-plasmid had to be carried out.
Thurs 16th September
- Top10 CMR transformation was successful. Four overnight cultures of each Top10 XylE and Top10 CMR were set off with either M9 or LB medium respectively.
- Piotr: Did mini preps from bacteria with blunt ended Xyle-GFP fusion and did diagnostic using both:
- digest with XbaI and SpeI
- PCR reactions with the following primers added: HIS-GFP, XylE-Xba and the other sample with HIS-GFP and GFP-flag
The PCR reactions were not very conclusive on the gels but digests allowed to determine that colonies 1->5 seem to have the right sizes of DNA in them and on Friday they will be prepared to be sent off for sequencing