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From 2014.igem.org

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.

Make up 8 flasks of 100ml starter culture.
4 flasks were checked each time to take the OD readings.
OD 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

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

Week 2

Monday 30^th June

Make 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 our biobrick plasmid.

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 option 3a.

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 plates were made, 2 ampicillin and 2 chloramphenicol.

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

Results are shown below;

Biobrick	Concentration
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 ensuring 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 moved 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 with the 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 to select for the correct colonies.
Plates were checked on saturday AM and ...

Week 3

Monday 7^th July

Make 200ml LB Agarand 200ml LB Broth.

Make overnight cultures of Tlia and KerUS.

Make overnight cultures of 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.

Concentration of DNA from plasmid mini-prep.
TliA - 5.2ng/μl
KerUS - 3.4ng/μl

Produce glycerol stocks of the Tlia and KerUS cells to be kept as backup.

Make chemically competent MC1000.

Measure the OD600 of the MC1000 cells using spectrophotometer

Flask 1 - 0.803
Flask 2 - 0.624
Blank - 0.066

Put 30ml MC1000 into falcon tubes. We have 4 tubes of full MC1000, the 5th one is 21ml MC1000 and 9ml water, the 6th one is all water.

Wednesday 9^th July

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

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