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Our first day in the lab working on our project began by resuspending Killer Red experiment part DNA from the iGEM registry plates in 10μl of MQ water and left for 5 minutes. The T5 promoter (BBa_K592008), Double terminator (BBa_B0015), RBS (BBa_B0034), non codon optimized Killer Red (BBa_K1184000), Strong, Medium and Weak Constitutive Promoters (BBa_J23101,BBa_J23110,BBa_J23103) resuspended DNA was then transformed, 1μl of DNA was inoculated into DH5a competent cells and then the transformation protocol (1) was followed. We plated on the corresponding antibiotic and LB plates and left the plates overnight in the non-shaking 37℃ incubator.
The transformation of resuspended parts from the previous day were not completely successful. We did have growth of 1-2 colonies from the constitutive and T5 promoters, as a result we decided to overnight these colonies but also re-transform the DNA to produce more colonies for the remaining parts. We believe the transformation was unsuccessful due to the concentration of the resuspended DNA being too low so we transformed a higher volume of 5μl.
The transformations were successful with the higher volume of DNA, consequently made overnights (protocol 2) of these colonies at the end of the day. We were able to Mini prep (protocol 3) the promoters from the first transformations which had been overnighted the day before. The strength of the promoter produced a significant colour difference between each overnight culture; the strong promoter was red, medium was orange and the weak promoter formed a yellow culture. Today we also carried out the pJet blunt end protocol (4) to ligate our Killer Orange G-block DNA insert with plasmid backbone using an empty backbone as our control. Resuspension: 20μl elution buffer to give 50mg/μl (pJet). Working with 3:1 ratio, use 3μl of insert to 1μl of backbone Composition of Killer Orange solutions detailed in table below.
| Solution A (μl) | Solution B (μl) | |
|---|---|---|
| Reaction buffer | 10 | 10 |
| PCR product | 1 | 3 |
| pJet | 1 | 1 |
| Water | 7 | 5 |
| T4 DNA Ligase | 1 | 1 |
We transformed 5μl of KO_A and KO_B.
Made glycerol stocks of the transformed parts were made using 500μl glycerol and 500μl E. coli overnight culture. Mini preps were then made and stored in the freezer.
Q5 site-directed mutagenesis (protocol 5) was used to remove restriction site EcoRI from the pKD4 plasmid available in the lab for Lambda red genome integration. The primers ordered for the Q5 kit were resuspended according to the protocol and then diluted to achieve 10μM concentration. The template pKD4 plasmid DNA was also diluted as only 10ng were required for the entire reaction. The Q5 protocol (5) was followed as a two step PCR reaction. Extension was run for 105 secs (3 x 30sec per kbp as pKD4 is 3267 bp, add 15 sec for extra time) Transformed Q5 PCR products into DH5a E. coli.
Our first Q5 site-directed mutagenesis attempt was unsuccessful
yesterday and so we carried out the procedure again using a three step PCR reaction by changing round
3 to 70? for annealing.
An 0.8% agarose gel was made to check that the PCR product was inclusive of the correct size
pKD4 3267 bp plasmid. PCR picture 1 Q5 attached below shows a successful PCR product.
PICTURE?
Also today the Qubit was used to quantify the concentration of mini prep DNA. We followed the Qubit protocol (6).
Jack spent today carrying out a digestion and ligation protocol (7) to join the resuspended parts from the registry.
We are following a multi step process in which the first digestion and ligation is used to ligate the promoter and RBS
as one part and the Killer Red WT protein coding region with the terminator as a second part. These two initial parts must
then be ligated to form the entire promoter, RBS, coding region and terminator sequence. Today's work involved the initial
digestion and ligation using a tetracycline plasmid backbone. The PCR product produced was then transformed into DH5a
Yesterday's digest-ligation transformation did not work. We repeated the digestion with RFP-tetracycline plasmid from our supervisor and produced more transformation controls to validate our experimental procedure. Q5- site directed Mutagenesis The PCR and gel electrophoresis were successful yesterday. However, the PCR product was not successfully transformed into DH5. It was later discovered that this is due to the pKD4 plasmid being toxic to this strain of E.coli. All of the PCR product remaining was loaded onto the gel so the reaction had to be repeated. The second gel was successful so the PCR product was transformed into E.coli DH5a and incubated at 37℃ overnight. However this transformation was also unsuccessful due to toxicity of pKD4 to this E.coli strain.
The successful transformation of the ligation products promoter and RBS, Killer red WT and terminator were overnighted and then today the Mini-prep protocol (3) was followed to retrieve this DNA. Qubit was then used to find the concentrations. MYE media was also made for future mini stat experiments testing different types of media for continuous culture of kill switches. See protocol 7. Transforming the Q5 PCR product was continued today using a new KLD enzyme mix. Cas9 protein was resuspended using too much water from the 2016 distribution kit so the correct amount of resuspension was added to our left over 2015 distribution kit. This was then transformed into DH5a.
Digestion and ligation of promoter + RBS, and CDS + terminator was carried out to join together the next set of ligations to create the full part for non codon optimized Killer Red. The ligation product was then transformed.
The new cloning protocol MoClo (Modular Cloning) requires the use of the ccdB gene which is
toxic to all but one strain of E.coli. We are using this cloning method to create our codon optimized
Killer Red and Killer orange for characterisation and part for the registry.
An adjusted digestion and ligation protocol was followed to cut RFP from the template backbones with each antibiotic
resistance (Ampicillin = A, Chloramphenicol = C, Tetracycline = T and Kanamycin = K) and the ccdB gene from the pS797
plasmid by digestion using enzymes with EcoR1 and PstI restriction sites. These digestion products were then run on a
gel so as to separate the plasmids and RFP/ ccdB genes.
The Promega Wizard SV Gel and PCR Clean- Up system was used to extract and obtain the separated DNA from the gel
to be used for ligation. The ccdB gene was then ligated into each plasmid. See ligation protocol (7). Once the
ligation was complete, the product was transformed into the ccdB survival strain from invitrogen.
The ligation protocol temperatures were altered for incubation at 37℃ for 15 minutes
then heating at 65? for 20 minutes.
The Order they went in gel (hyperladder): A, T, K, C, 797, 797. COULD INCLUDE PICTURE OF GEL?
The ligated killer red parts were mini prepped and the single successful Cas9 colony was overnighted. Unsuccessful ccdB gene work was re-transformed as it was assumed that ampicillin and kanamycin backbones were plated onto the opposite plates as only the tetracycline and chloramphenicol plasmid backbones grew colonies. An OD growth curve was produced for positive controls of 3 backbones, A,C,K. Killer Red ligations were sent off for sequencing to check that we made the part in the correct order sequence.
Glycerol stock, mini prep and qubit of Cas9 part were completed. Another experiment involving optical density in LB and no salt broth were undertaken.
The ccdB plasmid backbones that were not successfully transformed were tried again. This required more ccdB gene to ligate with gel extracted DNA. However we were unsuccessful in amplifying enough PCR product of the gene because the digested pS797 plasmid was in too low concentration. This was determined by again qubitting the pS797 stocks, we will use the highest concentration for the next digestion. Successfully transformed pSB1T3 ccdB and pSB1C3 ccdB plasmid backbones were overnighted to be used for cloning
More mini prep and qubit was carried out.
The QuickChange (QC)Multi kit was used today instead of the Q5 kit which had not been successful.
This kit is different in that up to 5 sites can be mutated at once, we are using it to swap three
separate base pairs in one EcoR1 site and two Xbal sites within the pKD4 plasmid. It involves three
forward primers and three reverse primers being added to separate PCR reaction tubes. See Quick change multi protocol (8).
Also today we began using the MoClo cloning technique to make codon optimized Killer red and orange parts.
See protocol (9). The resulting PCR product was transformed into the S171 E.coli strain.
Although production of pSB1T3 ccdB and pSB1C3 were successful, the digestion and ligation of the pSB1_3 ccdB plasmids was carried out again from the beginning and transformed into the ccdB Survival Strain from Invitrogen. The QC multi kit was unsuccessful yesterday and so was repeated again today to remove multiple restriction sites in the pKD4 plasmid. Overnights were made of the cloned KO/KR in DH5a.
Glycerol stocks, mini preps and qubit was carried out on the Killer Orange and Killer red overnights in S171. Overnights for successfully transformed ccdB plasmid backbones and KO/KR in S171 and DH5a were made.
Glycerol stocks, mini prep and qubit of ccdB plasmid backbones as well as KO and KR from MoClo in S171 and DH5a strains were carried out before sending these for sequencing. The QC multi was carried out again and the controls showed it to be successful by turning blue.
The pJET protocol was followed for our terminator, promoter and RBS, these were used for our mini experiments on reverse GFP. Transformations were also carried out for pKD4, KO and KR. Overnights of pSB1C3 and wild type DH5a were made to be used in the mini stat tomorrow. Dan and supervisor Paul investigated the setup and control of the mini stat today ready to be used tomorrow for initial testing.
Today MoClo was used to begin cloning DNase and Lysozyme for our additional Kill switch tests. The mini stat was set up. Starting optical densities (OD) of the overnighted pSB1C3 was taken before it was inoculated into the mini stat chambers. More overnights were made of the KO and KR as well as reverse GFP pJET products in DH5a.
All overnights from yesterday grew successfully. However DNase and Lysozyme did not form colonies even though positive and negative controls were successful. The mini stat showed significant colour differences in the growth of the RFP plasmid in different types of LB media, no salt seems to be fluorescing less. Glycerol stock, mini prep and qubit of the overnighted cultures. The pJET reverse GFP products were sent for sequencing. The MoClo and transformations of DNase and lysozyme were repeated. Sequencing information for the Killer red and orange protein came back to confirm which mini preps contain the correct DNA product. Killer orange was transformed into inducible strain BL21DE3. Killer red will also need to be transformed. The mini stat pump was set to 7.5 rpm and switched on at 13:30. The effluent needles were set to 20 ml. The air was switched off at time 0 when initial measurements were taken once the cultures reached the same volume level. We need to ensure that each time the mini stat has been thoroughly autoclaved and sterilised.
Pablo began MoClo cloning on reverse GFP using the pJet parts. Qubits of these parts were carried out before MoClo was started to ascertain the exact volumes required according to the DNA part concentration. Transformations of KR3 into BL21DE3, DNase and lysozyme into DH5a. The OD of the continuous cultures in the mini stat were taken. The burettes were emptied and the flow rate was lowered to reduce the amount of effluence produced overnight. Killer orange in BL21DE3 was overnighted. Four flasks containing 50 ml LB were set up in the hood and wrapped in tin foil ready for antibiotic and KO inoculation tomorrow. Dan prepared overnights of the LB in the ministat against LB without antibiotic so as to see if the chloramphenicol was still functioning in our LB media to understand how long the antibiotic remains effective.
Dan's experiment showed that the chloramphenicol was still working after 1 day in the mini stat. However by day 2 all media was contaminated. Again transformations of DNase and Lysozyme were not successful. Three of the flasks were inoculated with KO and one with control pSB1C3 RFP, OD was measured every hour using the Tecan and Cuvette reader until optical density reached 0.4. At this OD IPTG was added to flasks B and C containing KO. The induced flasks were left wrapped in foil for maturation of the protein for 1 hour. After maturation 200μL of each culture A,B and C were spread plated.The control, induced culture B and non-induced culture A plates were placed in the light box for 1 hour while induced culture C's plate was left wrapped in foil on the bench. After the hour all plates were placed in the cold room for testing on monday. The transformed KR plate was also placed in the cold room to be overnighted on Monday.
The spread plates of KO still had live colonies concluding that the kill switch had not worked. A sample of cultures of KO in flask that had been left over the weekend was taken and the rest was poured into plates and again left in the light box for the entire day. The sample was tested using the FACS machine, this showed that the cells were still live and on the non induced culture A, KO protein had been produced because the T7 promoter is leaky. Killer Red was overnighted.
DNase and lysozyme were cloned again then transformed into DH5a. Killer red overnights were glycerol stocked, mini prepped and quibitted. Flasks were set up to test Killer red and killer orange again.
The transformations of DNase and Lysozyme were successful. The colonies were overnighted. The flasks prepared yesterday were inoculated with KO and KR, OD reading were taken till 0.29 on the tecan reader was reached. After this OD reading each was induced with 100μL of 0.1 M IPTG. A 1.5ml sample was taken from each culture, spun down and treated with bug zapper in preparation for an SDS page gel the next day. The cultures were then incubated in at 37? and 220rpm overnight. The DNase and Lysozyme colonies were overnighted.
Overnights of the DNase and Lysozyme were glycerol stocked mini prepped and qubitted. 5ml samples of the overnight KO and KR cultures were pipetted into 10ml falcon tubes and placed label down in the light box set to 3. An SDS page gel was performed using the samples taken previously and samples taken 20 hours after induction. Spread plates were made of all the samples under the light after 6hrs of exposure.
Spread plates were checked to find all had live colonies. Work will begin again on Monday.
Our Enzcheck lysozyme kit arrived so we began by making stock solutions and aliquoting these reagents to be stored for later work. We transformed Killer Orange, Killer Red and Lysozyme into BL21 DE3 E.coli and started competent cell prep of this strain. Later in the day overnights were made to be used for testing tomorrow for an SDS page gel, these included Killer Red BL21DE3 and wild type DH5a. The DNase was again cloned using the MoClo method and placed in the PCR machine overnight. We prepared a streak plate of DH5a Killer Orange to be sent to Glasgow iGEM team for a collaboration.
MoClo DNase was removed from the PCR machine and transformed into DH5a. In addition to this KR3 was also transformed into DH5a. Overnights were made of all transformations from yesterday as all were successful. The lysozyme horizontal gene transfer experiment was performed (see lysozyme HGT protocol) and left overnight in the PCR machine at 55?. 5ml samples were taken from the culture flasks of KR induced, KR not induced and WT DH5a and put into the cold room.
We inoculated flasks from yesterday with 0.5ml of inoculum. And grew them to an OD of 0.4 for KR and 0.7 for KO and induced with 0.1ml of 0.1M IPTG prepared today and left overnight 37? and 220 rpm. Dilutions of the 20 hr and 4hr induced killer red from 23/8/16 were prepared to 10-1,10-2,10-3, these were then exposed to white light for 7.5 hrs and then 0.2ml of each was spread plated. The incubated lysozyme was removed from the PCR machine, 3μL was transformed in the usual protocol, 3μL was incubated with with 200μL and 0.7ml of LB. The left over lysate was plated out as a control to see if the cells survived the reaction. The EnzChek standard curve was performed. Competent BL21 DE3 cells were made and put into the -80℃? freezer. An SDS page gel was performed on the 4 hr and 20 hr induced KR, and WT DH5a, it didn't show any real difference. Overnights of the successful DNase plates were made.
The plates prepared last night using the lysate the the HGT experiment were checked. Plated out lysate showed no colonies, the full transformation protocol showed colonies producing RFP, the incubation condition also showed colonies. This is an initial indication that HGT of DNA from a cell that has undergone lysis using production of lysozyme can in principle happen. More repeats of the experiment will be undertaken. The overnight cultures of KO BL21 DE3, KR BL21 DE3 and pSB1C3 RFP DH5a were used to make 4.5ml samples at a dilution factor of 10-3,10-4 and 10-5. One set of fifteen samples (see Light Box protocol for samples) was exposed to light in the light box. A duplicate set of samples was kept in the dark outside the light box. The temperature inside the light box was also taken. It was observed that the temperature in the box was around 37℃, and outside was not. Repeats of the experiment will include the dark samples being covered in foil and placed in the box with the exposed samples. Fluorescence and OD will be taken of all dilutions before and after exposure from now on. All samples were spread plated.
CFU's were counted on the spread plates from the light exposure experiment. Concentrations of the DNAse miniprep was measured so this can now be sent for sequencing. Glycerol stocks were made of the KR3 in DH5a and lysozyme in BL21 DE3
Cultures were prepared for the Killer Red and Killer Orange experiment to be run (See protocol). The ministat chambers were cleaned ready t o be autoclaved. DNAse was transformed and the miniprep was sent for sequencing. 5ml overnights were prepared for a repeat of the Killer Red, Killer Orange experiment. Overnights for the interlab were performed.
Cultures were started from the overnights of Killer Red and Killer Orange ready to be induced, the cultures induced yesterday, were diluted and put in the light box. Spread plates were performed for each sample after 6hrs of exposure.
KR and KO were diluted and exposed to light for 6hrs before being spread plated. The FACS data for interlab was completed. The overnight cultures of the constructs were grown from a starting OD of 0.02 for 6hrs then samples were run through the FACS machine. The data was analysed and sent to iGEM.
CFU's were counted, there were two anomalies RFP 10-3 in the dark and KO 10-5 not induced , this data will be discounted as it was an issue with the plates the colonies were grown on
The chambers and media containers for the ministat were prepared and autoclaved. A new configuration for the effluent was set up using 1 litre duran bottles instead of 50 ml burettes. This solves the practical problem of overflow. Overnights of RFP, KR,KO,WT and lysozyme.
Set up a ministat, had issues with one of the chambers most likely because of blockage from using water when autoclaving the previous day. KR, RFP, WT were diluted and exposed to light for 5hrs (shorter time than previous because of some errors made during dilutions e.g putting LB with CAM and WT together). These were then spread plated and incubated overnight. Received -strain of E.coli insert- from glasgow.
CFU count of KR, BL21 (DE3) and RFP. WT grew in both conditions, showing RFP is phototoxic, but not to the same extent as KR or KO. Will repeat with WT as well as KO KR. Made LB plates. Issue with effluent bubbling over, fixed problem by moving effluent tubes higher to stop bubbles forming on top of eachother.
Counted CFU's for the KR KO experiment
Prepared overnights for KillerRed, KillerOrange and lysozyme samples.
KillerRed and KillerOrange and Lysozyme samples were grown up in 50 ml of media and induced. Overnights were made of the KillerRed and KillerOrange.
Ministat was inoculated with samples of KillerRed, KillerOrange and lysozyme in BL21 (DE3) and the batch phase of the culture started. KillerRed and KillerOrange experiment was performed
Ministat pump was switched on and the continuous culture started. CFU's were counted.
KillerRed and KillerOrange experiment was performed. Samples of each culture in the ministat were taken and glycerol stocked. Enzcheck assay was performed on the lysozyme samples at different dilution factors to determine what was optimal for the assay.
