Week 1

Monday 23^rd June

Make up 100ml of LB Broth and 100ml LB Agar.

Make an overnight starter culture of BBa_K258008 to test sterile technique.

Tuesday 24^th June

Generate Chemically competent E.Coli.

1. Make up 8 flasks of 100ml starter culture.
2. 4 flasks (A-D) were checked each time to take the OD600 readings.
3. OD600 readings to show when the cells are in exponential growth phase and so ready to be made competent.

   	A	B	C	D
   1.25h	0.077	0.078	0.082	0.096
   3.25h	0.567	0.674	0.714	0.809

4. Aliquot into 24 falcon tubes. The cells will need to be spun down in 2 rounds.
5. 72 eppendorf tubes will be produced, labelled ch. comp, dh5alpha.
6. Store at -80°C.

Week 2

Monday 30^th June

Make 5mL overnight starter cultures of the following;

Tube	BioBrick	Antibiotic
1	BBa_K258008	Chloramphenicol
2	BBa_K215107	Tetracycline
3	BBa_K861061	Chloramphenicol
4	BBa_K215002	Ampicillin
5	BBa_K1149021	Chloramphenicol
6	BBa_K861060	Chloramphenicol

5μl of an appropriate antibiotic was added to each culture to allow selection for cells containing biobrick plasmids.

Tuesday 1^st July

Conduct 6 mini-preps, one for each of the biobrick cultures, to produce 6 Eppendorfs containing purified DNA.

Wednesday 2^nd July

Run a DNA gel using the purified DNA collected yesterday to check DNA is pure.

Use an 8 well comb to produce the gel.
The table below shows the placement of each DNA sample within the Gel.

Well	Biobrick	Part Function
1	1kb gene ruler
2	BBa_K1149021	Promoter, RBS, Lipase
3	BBa_K258008	ABC transporter
4	BBa_K861060	pFadR promoter
5	BBa_K861061	pFadR Promoter + RFP
6	BBa_K215107	ABC transporter (strong)
7	BBa_K215002	pLAC +RBS +secretion signal + streptavidin binding tag

The gel was run at 100v for 45 minutes (due to time constrains). Below is the image taken of the Gel on the Gel dock.

4 LB agar plates were made, 2 ampicillin (100 μl) and 2 chloramphenicol (25 μl).

Nanodrop the DNA gained from the Mini-prep to generate concentrations of extracted DNA for all 6 biobricks.

Results are shown below;

Biobrick	Concentration (ng/μl)
BBa_K1149021	115.0
BBa_K258008	075.9
BBa_K861060	100.3
BBa_K861061	130.7
BBa_K215107	043.3
BBa_K215002	125.0

Transform cells to confirm competency

For this transformation We used the BioBricks BBa_K258008 and BBa_K215002 to check if the cells were competent.
Calculations are shown below;

BBa_K258008
75.9ng x X = 10ng x 100μl
X = 13.2μl

BBa_K215002
125.0ng x X = 10ng x 100μl
X = 8.0μl

Friday 4^th July

The linearised TliA and KerUS synthesised DNA is cloned into a pJet1.2 plasmid using blunt end ligation.

This ligation mix was then used to transform 3 eppendorfs of chemically competant cells (made on tuesday 25th);

Into eppendorf 1, pipette 5μl of the ligation mix containing TliA DNA.

Into eppendorf 2, pipette 5μl of the ligation mix containing KerUs DNA.

Into eppendorf 3, pipette 5μl of dH₂O

Incubated for 45 minutes due to time constraints.
Constructs plated on ampicillin LB agar plates to select for the correct colonies.
Plates were checked on saturday AM and positive colonies were observed

Week 3

Monday 7^th July

Make 200ml LB Agarand 200ml LB Broth.

Make overnight cultures of positive colonies of pJet 1.2 with TliA and KerUS.

Make overnight cultures of E. coli MC1000.

Tuesday 8^th July

Miniprep the Tlia and KerUS cultures to produce purified plasmid DNA.

Nanodrop the purified DNA to determine the concentration of DNA.

TliA - 5.2ng/μl
KerUS - 3.4ng/μl

Produce glycerol stocks of the Tlia and KerUS cells for storage (retain only if insert confirmed).

Make chemically competent MC1000.

Measure the OD600 of the MC1000 cells using spectrophotometer

Flask 1 - 0.803
Flask 2 - 0.624
Blank - 0.066

Decant the flasks of MC1000 into 50ml falcon tubes. We have 4 tubes of full MC1000, the 5th one is 21ml MC1000 and 9ml water, the 6th one is all water -to ensure the centrifuge is balanced (weight checked too).

Make 10ml overnight cultures of pJet 1.2 containing TliA or KerUS

Wednesday 9^th July

Repeat yesterdays miniprep as it didn't yield enough DNA.

Began with two 10ml starter cultures, one of pJet 1.2 TliA and one of pJet 1.2 KerUS.
Decanted by hand into falcon tubes to give four 5ml cultures.

A Restriction Digest was carried out on the Tlia and KerUS minipreps, as well as the FadR vector that was miniprepped last week.
TliA and KerUS were cut with xbaI and pstI, whilst FadR was cut with speI and pstI.

Thursday 10^th July

Run Vector, TliA and KerUS DNA on a gel to ensure it has been cut correctly. Once confirmed the DNA was then extracted from the gel.

Add 12.5μl 5X loading buffer to the DNA samples (50μl each) - total of 62.5μl in each eppendorf tube.
Load each sample, using two wells each if necessary.
Run at 100V for 60 minutes.

Visualise the gel.

The bands for TliA and Vector DNA were the correct size.
The band for KerUS showed that the DNA had been cut incorrectly.

The two samples of DNA that have been cut correctly are now extracted from the gel.

The gel extraction was then nanodropped to determine DNA concentration.

Vector: 14.3ng/μL
Tlia: 190.9ng/μL

A ligation reaction was carried out using the following amounts of reactants;

3.5 μl vector
0.2 μl lipase
2 μl buffer
1 μl ligase
13.3 μl dH2O

leave for 10 minutes at room temperature

Add 5μl ligation reaction mix to 200μl DH5α and transform.

NOTE: did not work -no positve colonies following antibiotic resistance selection on an LB agar plate
We also did some work to help us characterise the pFadR promoter.

Due to a mistake over biobrick numbers, our overnight contained pFadR without RFP, and so we used the day to test our experimental procedures.

Day cultures were made.

1 x 5ml of empty DH5α
3 x 5ml of DH5α containing BBa_K861060
Day cultures were placed in the incubator at 37°C, 170rpm for 3 hours

Lipid stocks were made using Triton to solubilise.

12 samples were made to be assayed;

1: 5ml DH5α + 0% lipid
2: 5ml DH5α + 1.0% olive oil
3: 5ml DH5α + 1.0% oleic acid
4: 5ml DH5α + BBa_K861060 + 0% lipid
5: 5ml DH5α + BBa_K861060 + 0.1% olive oil
6: 5ml DH5α + BBa_K861060 + 1.0% olive oil
7: 5ml DH5α + BBa_K861060 + 0.1% oleic acid
8: 5ml DH5α + BBa_K861060 + 1.0% oleic acid
9: 5ml LB + 1% olive oil
10: 5ml LB + 1% olive oil + chloramphenicol
11: 5ml LB + 1% oleic acid
12: 5ml LB + 1% oleic acid + chloramphenicol

Samples were incubated at 37°c

OD600 Readings were taken at hourly time points

100μl of each sample was pipetted into wells in a Transparent Greiner 96 microtitre plate.
As well as the 12 samples, 5 Control Wells (blanks) were created, without cells:

A: LB only
B: 0.1% Oleic Acid only
C: 1.0% Oleic Acid only
D: 0.1% Olive Oil only
E: 1.0% Olive Oil only

How to use the Plate Reader

Turn on computer
Select ...
Select open plate icon
Place microtitre plate into cassette holder
Select close plate icon
Open lid of plate reader
Twist wheels - for OD select white and red wires, for fluorescence select yellow and blue wires
Select spanner icon on program to choose between OD and fluorescence
Select Measure icon
Edit ….
Select Excel icon
Open A600 for OD, RFP Charlotte for Fluoresence
Select update icon
Scroll down to Table 2 to view results

Friday 11^th July

FadR assay analysis

Week 4

Monday 14^th July

Make overnight cultures.

DH5a + pSB3k3

DH5a + BBa_K861061

DH5a + BBa_K861060 x2

DH5a

DH5a + pJeT 1.2 TliA

DH5a + pJeT 1.2 KerUS

Tuesday 15^th July

Overnights of pJet 1.2 KerUS and TliA containing cells didn't grow, and so the time was used to make DNA stocks of BBa_K861060 and BBa_K861061.
Overnights were Miniprepped and then Nanodropped.
dH₂O was used instead of the elution buffer in the miniprep kit.

FadR - 80.6ng/μl
FadR - 75.9ng/μl
RFP - 50.3ng/μl

Make Overnights of pJet 1.2 + TliA and KerUS.

Wednesday 16^th July

Miniprep Tlia and KerUS cells.
Nanodrop

TliA - 522.0ng/μl
Kerus - 264.9ng/μl

Restriction Digests were made with TliA, KerUS and FadR containing plasmids.
The following amounts of DNA were cut with restriction enzymes and the final reaction mixture bought up to 50μl.

2μl Tlia
4μl KerUS
12.5μl FadR

Run a Gel to seperate out the restriction fragments.
Do a Gel extraction on the bands which are the correct size. The DNA was eluted into dH₂O. Nanodrop

TliA - 5.7ng/μl
KerUS - 7.0ng/μl
FadR - 5.7ng/μl

A Ligation reaction was set up containing either cut TliA or KerUS and FadR vector.
After an hour, the reaction mix was added to chemically competent DH5α which were transformed and plated on antibiotic containing LB agar plates.
Plates were incubated overnight at 37°C

Thursday 17^th July

No positve colonies from the TliA and KerUS with FadR vector transformation

Day cultures of BBa_861060, BBa_861061 and pSB3k3 were made.
Once grown (leave for around 5 hours), 10μl of these cells were then plated onto either 25μl 1% Oleic Acid, 25μl 1% Olive Oil or 25μl LB agar plates.
Cells were left in the incubator overnight.

Thursday 17^th July

No zone of clearing observed on olive oil containing plates

Week 5

Tecan data A second run was done on the Tecan. Three different cells were used;

BBa_k861060
BBa_k861061
pSB3k3

Each of these cells were added to solutions containing 0.5% Olive oil, 1% Olive oil and 2% Olive oil. Solutions without cells added were also used as a control.
200μl of each solution was pipetted into a 96 well plate with a clear bottom and white wells.
All unused wells were filled with LB to prevent evaporation of samples in the central wells. Fluorescence Microscopy

Set up overnights of the following;

PSB3K3
pSB3K3 + 1% Olive Oil
pSB3K3 + 1% Oleic Acid
BBa_K861060
BBa_K861060 + 1% Olive Oil
BBa_K861060 + 1% Oleic Acid

Add 10μl overnight cultures to microscope slide cover slips, allow to dry.
add 10μl 4% paraformaldehyde, dry for 30 minutes.
Wash 3 x with PBS buffer. Apply 10μl Prolong Gold Antifade Mountant with DAPI (Life Technologies). Fix to microscope slide, leave to dry overnight.
Use fluorescence Microscope to visualise cells with DAPI and RFP suitable channels. Maintain the same exposure time between images so they are comparable.

Following fresh ligation and transformation positive colonies were achieved for lipase with fadR responsive promoter in vector. Folllowing restriction digestion with xbaI and pst1. Negative result.

Overnights of BBa_k861060, BBa_k861061 x2, TliA x2, PSB3K3 pellets frozen for minipreps later

Week 6

Monday 28^th July

Miniprep of overnights from Friday (BBa_k861060, BBa_k861061 x2, TliA x2, PSB3K3)

Plasmid DNA stored in -20

Produce 6 flasks of LB agar.

Overnights of the following;

KerUS
TliA
BBa_k823017
PSB3k3

Tuesday 29^th July

Make up new stocks of soluble Olive oil (in Triton).

Make up 8 of agar plates with tributyrin in order to test lipase activity:

Differing concentrations of Tributyrin was added to either full strength or dilute LB agar. Any Olive oil was added to the empty plate and swirled to mix.

0.5% tributyrin
0.5% tributyrin + 2% Olive Oil
1% tributyrin
1% tributyrin + 2% Olive Oil

Make M9 Media
Too 800ml distilled H₂O add;

44g of Na2.2H2O
15g of KH2.H2PO4
2.5g of NaCl
5g of NH4Cl

Pour 800ml of distilled H₂O into 1L measuring cylinder
Disolve with magnetic flea and stirrer
Transfer media into 2 durans and autoclave.

To 10mL water add either;

1.2g of MgSO4
1.1g of CaCl2

Shake/invert until salts dissolve
Transfer into conical flasks, cover with cotton wool and foil, autoclave.

Make Glycerol stocks of pJet 1.2 KerUS, pJet 1.2 TliA, PSB3K3, BBa_K823017.

Made 6 LB Agar stocks.

Nanodrop of pJet 1.2 TliA and pJet 1.2 Lipase, BBa_k861060
Restriction and digest of pJet 1.2 TliA and pJet 1.2 Lipase with BBa_k861060. Transform into DH5a.

Wednesday 30^th July

One positive colony for TliA in the FadR vector in DH5a -ovenight culture of this

Thursday 31^st July

Miniprep the potential TliA in the FadR vector.
Nanodrop 54ng/μl Restriction digest of potential TliA in the FadR vector with NdeI and EcoR1 to confirm - failed
Make glycerol stocks of overnights potential TliA in the FadR vector

Friday 1^st August

Restriction digest undertaken.
TliA and a construct thought to contain the gene for TliA in the FadR vector were restricted using Xba1, Nde1 and both. A gel of the restriction digest was made
The gel showed that the fadR plasmid did not contain the lipase.

Week 7

ABC transporters into cells Overnights of both ABC Transporters (BBa_K258008, BBa_K215107), T1SS signal-strep binding tag (BBa_K215002).
Miniprep the above plasmids and transform into stocks of chemically competant DH5a.
Grow overnights of positive colonies, make competant and store at -80 in preperation for the transformation of a lipase containing plasmid.
Cloning
Miniprep the pJet 1.2 TliA and promoters BBa_J23101, BBa_J23110 and BBa_J23100.
Elute into 50&#956 of dH₂0.
Nanodrop

TliA - 103ng/μl
BBa_J23101 - 278ng/μl
BBa_J23110 - 408ng/μl
BBa_J23100 - 380ng/μl

Carry out a Restriction digest on the TliA and all three promoter plasmids - already had digested kerUS.

Slight variation to usual protocol: After 2 hours, 3μl CIP was added to the promoter plasmids and then returned to the heat block for another hour.
After 2 hours, the lipase was removed from the heat block. Make a gel
Restriction digestion mixes were all run on a DNA agarose gel and the correct sized bands cut.
DNA was extracted from the gel.
The TliA and kerUS inserts were ligated into each of the three different promoter strength harbouring vectors.
The ligation was left in the fridge overnight.
Ligation mixes were transformed into NEB5a cells
Positive colonies were seen for TliA and kerUS in the BBa_J23110 containing vectors
Colony PCR confirmed that TliA and kerUS had successfully been cloned into the BBa_J23110 vector
These colonies were grown in liquid culture and -80°C stocks were created

Tecan run with minimal media
DH5a plus the following plasmids were used for the tecan runs

FadR-Rfp
FadR
J23110

2 x 50ml falcon tubes of M9 media was made

35ml of dH₂O was added from duran
10ml of M9 salts was added from duran
2ml of casamino acids was added from falcon tube
100µl of MgSO₄ was added from 50ml conical flask
1ml of 20% glucose solution was added to one falcon tube, 1ml of dH₂O was added to the other
5µl of CaCl₂ was added from 50ml conical flask
topped up with dH₂O to make 50ml solution

Lipid was added to the M9 Solution
Total volume created = 4ml, in 6ml bijou tubes
Since the Olive oil:Triton X-100 ratio was 1:2, the following concentrations were made:

Solution	1%	2%	3%	4%
M9 Media	4.0ml	3.88ml	3.76ml	3.52ml
Olive oil:Triton	0μl	120μl	240μl	480μl

The 4.0% stock was made from the falcon tube without glucose added as a supplementary carbon source.
the 0.0%, 1.0% and 2.0% were made from the falcon tube with glucose added as a supplementary carbon source

Transformed cells & antibiotics were added to all 4 concentrations of M9/Lipid media.

96-Well plate was made.
200μl of each solution containing the 3 different promoters were pipetted into 4 wells to create 4 technical repeats.
200μl of each solution devoid of cells were pipetted into 3 wells in the region of the plate (B-G, 10-11)

Week 9

Monday 18^th August

Make overnights of kerUS and BBa_J23110

Tuesday 19^th August

Make 4 day cultures;

5ml KerUS + Nail
5ml KerUS + Hair
5ml BBa_J23110 + Nail
5ml BBa_J23110 + Hair

These were left in the shaking incubator for the cells to grow and produce and secrete keratinase.

Autoclave hair, nail and feather samples to sterilise. This was done in glass beakers with foil 'lids'.

Restreak KerUS and BBa_J23110.

Make overnights of kerUS and BBa_J23110.

Wednesday 20^th August

Overnights of BL21.

Thursday 21^st August

Make 2 5ml day cultures of BL21.
Allow around 3 hours for them to reach the exponential growth phase.
Take an OD reading
If the OD600 is over 0.6, use these innoculations to produce Chemically competant cells.

Miniprep KerUS BBa_J23110

Transform the chemically competant BL21 strain using 250ng of the purified KerUS BBa_J23110 DNA.

Plate the following;

100μl of control onto an antibiotic free plate.
100μl of control onto an ampicillin plate.
100μl of transformant onto an ampicillin plate.
Remaining 900μl spun down and resuspended in 100μl of LB and plated onto an ampicillin plate.

Transform TliA BBa_J23110 into stocks of ABC transporter (BBa_K258008) containing competent cells
Pour plates containing Lb Agar, Lb Agar containing 1% casein, Lb Agar containing 1% gelatin and Agar containing 1% gelatin.

Friday 22^nd August

Positive colonies of all transformations
Overnights of positive colonies of cells containing TliA/BBa_J23110/BBa_K258008, KerUs/BBa_J23110 and BBa_J23110

Saturday 23^nd August

Plate out KerUs containing overnights onto Casein, Gelatin and Gelatin LB agar plates. BBa_J23110 was used as a negative control. Staphylococcus aureus was used as a positive control.
Incubate for 8 hours, then flood plates in saturated ammonium sulphate.

Week 10

Monday 25^th August

Make overnights of KerUS/BBa_J23110, tliA/BBa_J23110, tliA/BBa_J23110/BBa_K258008 and BBa_J23110.
Autoclave four 1 Litre conical flasks each containing 100ml of LB Broth.

Tuesday 26^th August

Innoculate each of the autoclaved flasks with 1ml of each of the overnights. Add the correct antibiotic to each.
Incubate at 30°C to promote protein expression.
Give 5 or 6 hours for the cells to reach exponential growth phase and produce an adequate amount of protein to assay.

After 6 hours, samples were taken for analysis by protein gel.

Into 4 eppendorfs, pipette 1.5 ml of each innoculation.
These are spun down and the supernatant removed into a second tube. Following the addition of 150μl trichloroacetic acid (TCA) and kept in the 4 degree overnight.

Into 4 more eppendorfs, pipette 0.5ml of each innoculation.
These are spun down, the supernatant poured away, the pellet labelled and kept in the -20 degree.

Remaining culture was centrifuged, sterile filtered and loaded into Vivaspin 20 10,000 MWCO columns (GE Life Science) (tliA without exporter was excluded) add 5ml of culture supernatant. These were spun down at 3,500 rpm for 8 minutes.
Supernatant that had passed through the column was discarded, and more supernatant was added into the top of the column. This was repeated until around 15ml of supernatant had passed through the column.
Around 1ml of concentrated supernatant was then removed from the top of the column.

Overnight liquid cultures of cells containing kerUS (no promoter), tliA/BBa_J23110 and pSB1C3

Wednesday 27^th August

To prepare the protein sample to be loaded onto a gel

To the cell pellet add 100μl 2X SDS buffer. Resuspend the cells in this solution.
Centrifuge the 1.5ml of supernatant/TCA mix for 15 minutes at 13,300rpm, remove supernatant, resuspend pellet in 900μl acetone, repeat centrifugation step, remove supernatant, allow remaining acetone to evaporate then add 50μl of SDS buffer (a ratio of ~1:20)
To the 1ml concentrated supernatant, add 1ml of SDS buffer (a ratio of 1:1)
Place all of these samples into a heat block at 95°C for 5 minutes. The cell pellet may need to be vortexed and left for an additional 5 minutes.

Make a 12% SDS-PAGE gel. Load 5μl cell lysate samples and 15μl supernatant (concentrated or non-concentrated) onto the gel
Stain the SDS-PAGE gel with InstantBlue (Expedeon) overnight

Miniprep plasmids from overnight cultures, set up restriction digests with EcoRI and PstI (add CIP to the acceptor vector). Run donor mixes on a DNA gel and isolate DNA from bands of correct size
Ligate isolated cut kerUS and tliA/J23110 with cut pSB1C3 overnight

Thursday 28^th August

Transform both ligation mixes into NEB5a and plate on chloramphenicol containing LB agar plates

Friday 29^th August

Positive colonies on both LB agar plates. Colony PCR to confirm insertion of kerUS or tliA/J23110.
Overnight liquid cultures of a positive colony from each.

Saturday 30^th August

Miniprep overnight cultures. Send some for sequencing with sequencing primers. Once confirmed send on to the BioBricks Registry