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Chronological notebook

• 27th of June:

B. subtilis (strain: 168) culture inoculated into 10 ml LB medium. Overnight incubation in 37 deg.C

Preparation of metal ion stock solutions: Ni - 0.1M; Zn - 0.1M; Fe - 0.5M; Cu - 0.5M; Co - 0.1M; Mn - 1M (Aleksandra, Harry and Jan)

• 28th of June:

0.5 ml units of O/N culture diluted to 50ml (x100 dilution in LB medium). 3 ml of diluted culture aliquots poured into universal bottles (enables adequate aeration of the culture). Those were inoculated with 30 ul of differently concentrated metal ion solutions giving final concentrations of:

Ni - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Zn - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Fe - 5mM; 3.75mM; 2.5mM and 1.25mM; Cu - 5mM; 3.75mM; 2.5mM and 1.25mM; Co - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Mn - 10mM; 7.5mM; 5mM; 2.5mM; 1.25mM and 0.625mM

0.4 ml of O/N culture diluted to 40ml (x100 dilution in 2xLB medium). 3 ml of diluted culture aliquots poured into universal bottles (enables adequate aeration of the culture). Those were inoculated with 1ml of differently concentrated ethanol solutions giving final concentrations of:

2%; 4%; 6%; 8%; 10%; 15%; 20% and 25%

The initial Absorbance readings at OD600 for diluted cultures were taken. Following 7h. incubation in 37 deg.C absorbance of each sample was measured and compared to samples inoculated only with H2O (Kyle, Harry, Aleksandra, Dainius and Jan).

• 1st of July:

B. subtilis EtOH resistance assay was continued in a narrow concentration range (Jan).

E. coli (DH5alpha) cells were transformed using BioBricks required for EtOH production (Aleksandra).

O/N cultures of E. coli and B. subtilis were set up to continue with the experiments tomorrow (Harry).

• 2nd of July:

Transformation was successful. Four colonies were selected and propagated into 5ml LB bottles supplemented with adequate antibiotic (Jan). After 8h incubation in 37 deg. C cells were pelleted and plasmids were extracted using Qiagen MiniPrep Kit (Aleksandra). Concentration and quality of DNA was measured using NanoDrop.

E. coli cells plated for competitors were harvested and propagated into 50 ml LB medium. Following 4h incubation they were used to make competent cells for future transformations (Aleksandra and Jan).

B. subtilis resistance to heavy metals experiment was continued in a narrow metal ion concentration range (Harry).

• 3rd of July:

Since we propose using B. subtilis as a chassis for our waste water treatment bacteria, we first set about determining the concentration of different metal ions that the non genetically altered chassis can cope with. We initially observed cell density of cultures after 8 hours incubation with varying metal concentrations. Following this we recorded culture density every hour to obtain growth data for appropriate concentrations of metals, as indicated by the initial 8 hour cultures. These experiments continue... (Harry, Danius, Hugo)

• 4th of July:

The competent E. coli cells were tested by transforming them with parts for ethanol production. The negative control was performed to confirm that cells have no indigenous antibiotic resistance and that prepared antibiotic plates have antibacterial properties (Aleksandra and Jan).

Time-lapse measurements of B. subtilis growth in specified metal concentrations (based on previous data) were performed (Harry, Hugo, Dainius and Kyle)

Team started preparing poster (Kyle) and presentation (Dainius and Hugo) for the Young Synthetic Biologist meeting held in London over the next weekend: 12-14/07/2013

• 5th of July:

Outcome of the transformation was positive with the negative control showing no growth (as expected). Four colonies were collected and grown in 5ml LB supplemented with adequate antibiotic. Following 7h incubation the entire culture was pelleted and plasmid was purified using Qiagen MiniPrep Kit. NanoDrop was used to measure the DNA concentration (Harry and Jan)

Time-lapse measurements of B. subtilis growth in specified metal and ethanol concentrations (based on previous data)were performed (Harry, Hugo, Dainius and Jan).

• 8th of July:

Two PCR reactions were set up to clone out genes of interest from B. subtilis (Harry and Hugo; Dainius and Kyle).

The ethanol production part was combined with a promoter - overnight ligation(Jan)

• 9th of July:

The PCR product was run on the gel to confirm the presence of the desired product (Aleksandra, Dainius, Jan)

The ligation product was transformed into the competent E. coli cells. Those were plated on the KAN+IPTG+X-gal plates (Aleksandra, Jan).

B. subtilis genomic DNA was purified from the O/N culture for the use in the future PCR reaction (Harry)

• 10th of July:

DNA gel electrophoresis of the PCR product was performed once more due to inconclusive results from 9/07/2013 (Jan). Bands of expected size were observed. Additional PCR was set up to obtain more DNA (Harry and Hugo). Following gel electrophoresis resulting products were restricted using correct enzyme and an O/N ligation reaction was set-up (Aleksandra and Jan)

The transformation using the EdiBrick and ethanol production parts ligation product was successful - numerous blue colonies were obtained. Two colonies were selected and propagated into 5ml LB +KAN medium. Two more colonies (4 in total) were propagated onto LB+KAN+X-gal plates and incubated O/N in 37 deg.C (Aleksandra, Jan)

• 11th of July:

The ligation products of vector and two transcription factors amplified using PCR were transformed into E. coli and plated on LB+AMP+X-gal+IPTG plates (Harry and Weike)

• 15th of July:

The transformation done using the ligation product gave negative results. The restriction of SinR; Fur and AmpR vector was repeated. O/N ligation was set up using new restriction products (Harry, Weike and Jan)

Cells from four different plates of the EdiBrick_pET ligation were resuspended in 5 ml LB medium + KAN and grown in 37 deg.C. The plasmid was purified following 4 hours of incubation using Qiagen MiniPrep Kit (Aleksandra and Jan)

• 16th of July:

The DNA resulting from ligation was used for transformation of competent E. coli. Cells were plated on LB+AMP+IPTG (20 ul) plates (Weike and Hugo)

The EdiBrick_pET DNA was restricted using XbaI and PstI in CutSmart buffer. 2 h. incubation in 37 deg. C was used for restriction reaction.The resulting proucts were run on the gel (15ul product + 3ul loading dye). Three out of four DNA samples showed correct band sizes (Aleksandra).

Reagents required for ethanol assay were prepared and a sample data for 0; 0.5 mM and 1 mM were obtained in order to plot a standard curve. O/N cultures of E. coli with pET and EdiBrick_pET were set up in order to analyse those tomorrow (Aleksandra and Jan).

Timelapse analysis of B. subtilis growth in varying concentrations of three different heavy metals was performed (Harry and Dainius)

2xLB was prepared to perform the time-lapse analysis of B. subtilis growth in higher EtOh concentrations (Jan).

• 17th of July:

Due to negative results of ligation all DNA involved in the process was analysed using DNA gel electrophoresis to identify the faulty step (Kyle, Dainius and Hugo)

Supernatant of the pET and EdiBrick_pET O/N cultures was subjected to the ethanol assay. Due to encouraging results the pET plasmid was isolated and restricted using XbaI and PstI to confirm it definitely has the correct insert (Aleksandra and Jan)

• 18th of July:

B. subtilis growth in higher [EtOH] was analysed (Aleksandra and Jan)

PCR reaction was set up to obtain the DNA fragments required to perform deletions in the B. subtilis sporulation pathway (Dainius and Kyle)

PCR reaction for the MABEL based fusion of adhB and pdc was performed. Product was analysed on a gel. An O/N blunt-end ligation reaction was set up to obtain the plasmid with fusion proteins(Aleksandra and Jan)

Restriction of the Fur and SinR PCR products was performed using an enhanced protocol with an O/N incubation in 37 deg. C (Dainius)

• 19th of July:

Due to low amount of product obtained second PCR reaction was set up to amplify the DNA fragments required to perform deletions in the B. subtilis sporulation pathway (Dainius and Kyle)

The O/N restriction product was purified from the reaction mix. This was used for two separate ligation reactions using different ligant-to-vector ratios and [vactor] (Hugo and Dainius)

New GenBrick linkers were prepared to be tested over the next week (Aleksandra)

• 20th of July:

The ligation products were used for transformation of super-competent E. coli cells (Hugo and Dainius)

• 22nd of July:

The transformation result was positive. Four colonies per insert were selected and subjected to colony PCR. Moreover, those were propagated into 5ml LB + AMP cultures. Cultures were grown for 7 h and entire 5 ml was used for plasmid purification using the Qiagen MiniPrep kit. The resulting DNA was quantified using NanoDrop (Hugo and Dainius)

The MABEL blunt end ligation product was transformed into competent E. coli cells and plated on LB+KAN+IPTG+X-gal plates. Those were incubated in 37 deg. C O/N. Moreover, the purified linear MABEL product was run on the DNA electrophoresis gel to assess the quality of the large fragment (>7 kb) purification process when using Qiagen PCR product purification kit (Aleksandra and Jan)

• 23rd of July:

Cloned parts were excised from working vector and inserted into pSB1C3. A 2h restriction with XbaI/ PstI and an O/N ligation was performed to achieve that. Moreover, cloned parts were mixed with sequencing vectors so they are ready to be submitted to a sequencing facility tomorrow (Hugo and Dainius)

The transformation resulting from the blunt end ligation was successful. Six colonies were chosen and propagated into 5ml LB medium + KAN. Following 7h incubation in 37 deg. C the cultures were propagated into fresh 5ml LB + KAN + 2% lactose to induce expression of pdc_adhB fusion protein. The non fusion cultures were set up and induced at the same time to have a point of reference (Aleksandra and Jan)

E. coli JM109 cells were made chemically competent. E. coli and B. subtilis were patched onto fresh LB medium to be used in other experiments. Some CML plates were prepared (Aleksandra and Jan)

• 24th of July:

The ligation mix was used for transformation of competent E. coli cells. X-gal and IPTG were included in the plates (Hugo and Dainius)

A PCR reaction of the flanking regions required for deletion in the B. subtilis genome was performed (Kyle)

SDS-PAGE experiment was run to analyse the cell lysate from un-induced and induced E. coli bearing a plasmid coding for pET operon and pET operon fusion. The gel was left for staining O/N. The samples were also analysed for ethanol production (Aleksandra and Jan)

• 25th of July:

The transformation was successful. About half of the cells had inserts of interest in pSB1C3. Those were propagated into 5ml LB + CML. A MimiPrep was performed in the afternoon to obtain a pure DNA sample for sequencing (Hugo and Dainius)

SDS-PAGE was destined for an hour and photographed. De-staining was continued o/n (Aleksandra and Jan)

• 26th of July:

The pSB1C3 with Fur and SinR was purified from o/n cultures using Qiagem MiniPrep Kit. The DNA samples are ready for the submission to the Registry (Hugo and Dainius)

Three cultures of pET operon fusion showing correct band on the SDS-PAGE were subjected to Qiagen MiniPrep kit. The purified plasmid was restricted using PstI and XbaI and used for ligation (4 deg. C incubation) with the pSB1C3 - RFP restriction product. All restriction products were analysed on an agarose gel (Aleksandra and Jan)

• 29th of July:

The pET fusion_pSB1C3 ligation product was used for transformation of competent E. coli cells. Cells were plated on LB+CML+X-gal+IPTG plates to enable blue- red screening (Aleksandra and Jan)

A set of eight O/N E. coli cultures was set up to test influence of varying concentrations of lactose, xylose and glucose on ethanol production (Aleksandra and Jan)

GenBrick assemblies prepared last week were used for transformation of competent E. coli cells (Hugo and Weike)

B. subtilis wild strain was recovered from a freeze-dried ampule and plated on nutrient and LB agar (Kyle)

• 30th of July:

Ethanol production in media containing different sugars was analysed using an ethanol assay (Aleksandra and Jan)

Colonies resulting from the transformation were picked and propagated into 5ml LB + CML medium in order to purify plasmid with insert and pSB1C3-RFP tomorrow (Aleksandra)

• 31st of July:

O/N cultures expressing the Reporter genes for GenBrick assembly and pET fusion in pSB1C3 were subjected to Qiagen MiniPrep Kit in order to purify plasmids. Obtained plasmids are ready for the submission to the Registry (Aleksandra and Jan)

New reagent: Nitro Blue Tetrazolium was tested as a component of ethanol assay. Standard measurements were taken at both 530 and 570 nm in order to evaluate the suitable wavelength (Aleksandra and Jan)

Following the protocol for competent Bacillus subtillis cells taken from http://openwetware.org/wiki/Cfrench:BacTrans2 . Made some caramel minimal medium, the recipe is uncertain at the moment. The new B. subtilis was put to grow O/N in it. (Dainius and Kyle)

PCR and Gel of SinI and SinR (Hugo)

• 1st of August:

Ethanol production in media containing different sugars was analysed using an alternative ethanol assay with NBT as a dye (Jan)

GenBrick linkers with and without Fur box were phosphorylated and annealed. An O/N reaction of fusing the linkers to parts required for Fur box characterisation was set up (Hugo and Jan).

The caramel minimal medium is not toxic to B. subtilis cells. So i continued working with it and aliquoted the overnight culture into fresh medium for 3 hours, then added starvation medium for 3 hours, aliquoted 0.3ml into ependorfs and put into -80C. (Dainius)

• 2nd of August:

Dainius and Kyle made some more caramel minimal medium. Great success! 1 part salt, 1 part sugar, 1 part love, 200ml of DI water, try to measure pH and fail, put to autoclave for 2hours, serve hot.

God gave us some real minimal salts, so we worked with that instead, following the protocol used with caramel medium on 31st July. (Dainius and Kyle)

PCR purification of fusions from yesterday were performed. Four assemblies were made and used for E. coli transformations on CML plates. peace. (Hugo)

• 3rd of August:

Three colonies of 3 different assemblies from yesterday were put in LB broth. The rest of the competent transformed cells were concentrated and plated on CML plates. Will it work? Will it not? The suspense is great. Tune in tomorrow to see if the plates grew. (Hugo)

Meanwhile biofilm forming Bacillus subtilis competent cells were made and put in -80C, ready for action. (Dainius)

• 5th of August:

Competent biofilm forming B. subtilis competent cells were transformed with David's kanamycin resistance plasmid and plated out. (Dainius and Kyle)

Some transformations were made with GFP and RFP. (Hugo)

A native PAGE gel was casted for functional analysis of pET and pET fusion (Aleksandra)

A PCR was run and analysed on a 2% agarose gel to establish the size difference in the spacing between fused and non-fused pET operon genes (Jan)

Set of nine O/N cultures was set up. Those are both induced and not induced for ethanol production parts expression. The cultures will be analysed on a native PAGE tomorrow (Jan)

• 6th of August:

A PCR was run and analysed on a 2% agarose gel to establish the size difference in the spacing between fused and non-fused pET operon genes. This time a different DNA polymerase and highier primer concentrations were used. A sucesfull reaction indicated bands of size corresponding to fusion and non-fusion genes on the gel (Jan)

Two native PAGEs were run. Firs one was subjected to o/n comassie staining. The second gel was subjected to functional staining for AdhB activity (Aleksandra and Jan)

A PCR was run to mutagenise a shuttle vector using GenBrick based EMMA technique. The aim is to create a plasmid which could be used in both E. coli and B. subtilis and would be BioBrick compatible (Harry and Gavin)

A transformation of RFP and GFP parts required for GenBrick assembly characterising the Fur binding sequence was plated out (Hugo)

Nothing grew on the kanamycin plates with Bacillus subtilis. We will try a new method tomorrow. (Dainius and Kyle).

• 7th of August:

To obtain even more GenBrick reporter parts two more transformations were performed (Hugo).

A PCR was run to amplify genes coding for SinR and Fur transcription factors. Set of new primers was used with both BioBrick and GenBrick standard overhangs. PCR products were analysed on a gel and restricted using PstI and XbaI. An O/N ligation was set up which would insert those into pSB1C3 (Jan).

Two native PAGE were casted to be used in functional analysis of AdhB (Aleksandra)

Set of 12 E. coli o/n cultures with appropriate additives were set up. Those will be analysed tomorrow for both ethanol content and adhB activity, using native PAGE (Aleksandra and Jan)

Three more PCRs were run to mutagenise a shuttle vector using EMMA technique. All products were analysed on a gel. Fragments of expected size were observed (Harry and Gavin)

Competent E. coli cells were transformed with a vector on which new shuttle will be based in order to obtain more DNA to work with (Harry and Gavin)

A new method for transforming B. subtilis was used for introducing kanamycin resistance (Yasbin method). (Dainius)

• 8th of August:

Proteins were extracted from a set of 12 o/n cultures and prepared for an analysis on a native PAGE. Tween 20 detergent was included to increase amount of protein in the sample (Aleksandra)

A PCR was run to amplify entD; entE; entF genes and flanking region of SinR gene. PCR products were analysed on a gel and stored in -20 deg. C for purification and restriction tomorrow (Kyle and Jan)

O/N ligation product was used for transformation of competent E. coli cells and plated on CML+IPTG plates (Jan)

PCR products required for construction of B. subtilis - E. coli shuttle vector were purified using Qiagen PCR product purification kit (Harry and Gavin)

Failed. (Dainius)

• 9th of August:

Trying it again, but also with B. subtilis 168. (Dainius)

• 12th of August:

• 13th of August:

• 14th of August:

• 15th of August:

• 16th of August:

Recipes (or protocols if you prefer so)

• 2xLB medium:

Tryptone, 20g Yeast extract, 10g NaCl, 10g (when making more medium add 20g) Dissolve in 800 ml dH2O, adjust pH to 7.0 with NaOH/HCl, adjust volume to 1 litre and autoclave.

NOTE: Use this medium when diluting the culture with high volumes. If possible try to use same volume of 2xLB and the solution you want to add to it as B. subtilis does not grow really well in highly concentrated LB.

• Preparation of competent E. coli cells:

TSS buffer is required for this process. 24 ml TSS:

-17 ml LB -5 ml 40% w/v PEG 3350 -1 ml 1 M MgCl2 filter sterylise the mix above and add: -1 ml DMSO

Procedure:

Propagate E. coli from plate into 50 ml of LB medium. Grow until OD600 of about 0.2 to 0.5 is obtained. Pellet 1 ml of culture for each eppendorf of competent cells. Remove supernatant and add 100 ul of ice-cold TSS buffer. Incubate with TSS for 30 min on ice and store in -80 deg.C

• Transformation of competent E. coli cells:

1.Use 100ul aliquots of competent E. coli cells from the -80 deg. C freezer.

2.Place cells directly on ice. Try to limit the exposure to heat (i.e. do not touch the bottom of the eppendorf)

3.Add 2 ul of plasmid DNA to the cells and incubate on ice for 30 - 60 minutes.

4.Heat shock the cells in 42 deg. C for 90 seconds (exact timing)

5.After heat shock place the cells on ice for additional 90 seconds.

6.Add 900 ul of LB medium to the cells.

7.Grow in 37 deg. C shaking for 45 - 60 minutes.

8.Plate 100 ul of cells on a plate with appropriate antibiotic.

• Ethanol assay:

Buffer used in the assay: 50 mM tris-HCl pH= 7.5 (0.118g tris base + 0.635g tris.HCl in 100 ml of H2O)

Following solutions were prepared in the buffer:

- yeast ADH: 5 mg/ ml

- NAD+: 20 mM (13.3 mg/ ml)

- PMS: 20 mM (6.1 mg/ ml)

- INTV: 50 mM (25.3 mg/ ml) --> INTV has low solubility; can add few drops of DMSO to dissolve.

- ethanol: 30 mM (1.75 ul/ ml) --> required for the standard absorbance curve

Procedure:

Into 1ml of buffer add:

- 5ul yeast ADH

- 1 ul NAD+

- 1 ul PMS

- 20 ul INTV

mix well

Add 33 ul of the standard ethanol solution (30 mM) and 33 ul of diluted (DF=2) ethanol solution to obtain 1mM and 0.5mM standard responses respectively. The 0 mM EtOH response is obtained by adding 33 ul of H2O. Mix well after addition of the ethanol.

Incubate the assay for 15 min in room temperature. Following that take the OD500 readings which will correspond to the ethanol concentration. You can use H2O as a blank.

To evaluate the levels of EtOH in your bacterial medium spin down the cells from O/N culture and use 33 ul of supernatant as a substrate for the assay. The readings should be taken following exactly the same incubation period as ethanol standard. Make a fresh standard each day you want to analyse bacterial culture.

• Preparation of SDS-PAGE:

12% resolving gel:

H2O: 6.6 ml 30% acrylamide mix: 8.0 ml 1.5 M Tris (pH 8.8): 5.0 ml 10% SDS: 0.2 ml 10% ammonium persulphate: 0.2 ml TEMED: 0.008 ml

5% separating gel:

H2O: 3.4 ml 30% acrylamide mix: 0.83 ml 1.5 M Tris (pH 6.8): 0.63 ml 10% SDS: 0.05 ml 10% ammonium persulphate: 0.05 ml TEMED: 0.005 ml

5 x Tris-glycine electrophoresis buffer (running)

94 g Glycine and 15.1 g Tris base ---> dissolve in 900 ml of H20

add 50 ml of SDS (10% w/v)

make up to 1000 ml