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Week One

Dear Diary

This week we started lab work. We started by growing some Colis, making some amp-plates, making RIP DNA, making competent cells and trying to transform biobricks.

Day One - Preparation.

On the first day we streaked a strain of E. Coli called Top10 onto 5 LA plates (LA = LB medium with Agarose, LB = Luria-Bertani broth [http://en.wikipedia.org/wiki/Lysogeny_broth]). The plates were incubated at 37 degrees C overnight. [http://en.wikipedia.org/wiki/Streaking_(microbiology) Streaking] was done as shown on the picture [PICTURE FROM NOTEBOOK].

We also made LA plates containing ampicillin as our selection antibiotic. We decided to use an ampicillin plate-concentration of 50 μg/mL based on reported ampicillin resistance of our biobrick containing plasmids. We later found out that the plasmids provide sufficient ampicillin resistance for harsher selection with 100 μg/mL[#]. How to make 50 μg/mL plates.

Day Two - DNA creation and purification.

Today we started creating our RIP DNA. We had previously ordered primers with an overlap in order to make RIP DNA via [http://en.wikipedia.org/wiki/Pcr PCR]. In order to achieve a primer-stock concentration of 100 μM we added diH2O(de-ionized, autoclaved water) to our raw primers:

Name	Added Water	Description
Igem-1	187μL	RIP with export sequence (sRIP)
Igem-2	200μL	RIP with export sequence (sRIP)
Igem-3	301μL	RIP
Igem-4	269μL	RIP

Since we dont need all the DNA at once, we diluted the primer solutions to 20 μM, by adding 40 μL of diH2O to 10 μL of primer-stock.

We did PCR with our 20 μM solution of primer using this protocol, due to a shortage of Pfx-enzyme, we used Pfu enzyme instead and didn't add MgSO4 (which the Pfu-buffer already contains). We also elongated at 70 degrees C for 2 minutes. We made 4 tubes for both sRIP and RIP for extra safety.

Inspection of yesterdays platestreakings. We observed growth on all 5 LA plates. Tertiary growth on several plates. 1 plate has been streaked too deeply, it's hard to see colonies.

Post-PCR gel preparation. 4 μL of diH2O and 1μL of loading buffer (containing a colouring agent) was added to 5 μL of PCR product. Process was repeated for all 8 tubes. The solution was applied to a 1,5 % (mass/volume, 4,5g agarose in 300mL of diH2O) agarose gel og run for about 15 minutes, or until the bands reach halfway of the gel. Technically the solution is applied to the gel by inserting the tip of a pipette into the appropriate slot and releasing the material so that it stays in the slot, remember to apply a size-appropriate DNA ladder to a slot for size-determination of samples. [Gel-billede 1].

Tube-inoculation. We applied a colony from our LA-streakings to LB liquid medium and grew it overnight at 37 degress C and shaking. These cells are later made competent.

Gel purification of PCR product. Solution for gel-electroforesis was prepared as described earlier except, we now prepared 50 μL, by adding 5 μL of loading buffer to 45 μL of PCR-product. applying the solution to deeper, wider slots, run the gel as before, excise the bands, and weighing them.

Name:	sRIP-1	sRIP-2	sRIP-3	sRIP-4	RIP-1	RIP-2	RIP-3	RIP-4
Weight	390mg	420mg	520mg	560mg	719mg	600mg	450mg	430mg

We now followed a purification protocol, complete with kit. To the eppendorf-tubes containing the excised bands we added 1 μL of capture buffer per mg gel, heated to 60 degrees C and vortexed until the bands had been dissolved. From the tubes we transferred 600 μL to microspin tubes (w/ filter) and centrifuged for 30 seconds at 14500 RPM to capture the DNA in the filter. Flowthrough was discarded. From here on, as protocol describes.

Day Three - Competence and transformation.

We started the day by making competent cells according to this protocol. We measured OD550 (optical density at 550nm wavelength) as:

Time	#1	#2
09:00	0,036	0,040
10:15	0,08	0,115
10:45	0,167	0,253

We used 1mL pure LB medium as reference.

The PCR product we purified yesterday was tested in the same way as the product was before purification. [Gel-billede 2].

Using the competent cells from earlier and this protocol we attempted to transform our Colis with biobrick plasmids B0015, B0034, R0011, J23100, pSB1A3 (containing p1010) and 1 negative control (without plasmid). 1 hour incubation time on 37 degrees C.

Week 2

Dear Diary

This week we started working on the Wiki and continued our labwork. In the lab we had to start our transformation over, since we didn't have any usable growth in last weeks transformed cells. In the second round of transformation we added a second protocol (electroporation) and made minor changes to the classic protocol.

Day One - Starting over.

Transformation control. Sadly, we observed no growth on most of the plates from our first transformation. Only the plates containing biobrick B0015 (the double terminator in plasmid pSB1AK3) showed clumpy growth [images], 3 out of 4 plates (both 5 μL and one 2 μL) showed growth. One possible explanation attributed these observations to an uneven distribution of ampicillin in the medium, most likely though is that our competent cells werent quite so competent. Nevertheless we needed to start over with the transformation.

Amp preparation. We added 5 mL of diH2O to NEW powder ampicillin and vortexed to dissolve, creating an amp concentration of 100mg/mL. We transferred 1mL to each of three eppendorf-tubes. From the tubes we transferred 0,25 mL to ~500mL of LA medium, creating a ~1:2000 concentration gradient, with a medium concentration of amp = 50 μg/mL, to evenly distribute the amp in the medium it was shaken and rotated vigourosly.

Due to a mysterious lack of liquid in well 23L(B0015, double terminator) we decided to dilute all open wells (2M, 3K, 6G, 18C, 23L) with 2 μL of diH2O.

Electroporation. We made 7 samples, 5 parts, one negative and one positive control. protocol. We used 0,2 cm cuvettes. The tubes contained

3K
6G
2M
23L
18C
Positive control
Negative control.

From the seven sampletubes we made 7 LA+AMP plates and 1 LA plate(for the negative control).

Transformation and growth. We made 8 Plates fra each set of transvectorisation (transformation and electroporation), Using this naming scheme: "LA(+AMP) Well# (Electro OR transformation)". For our positive control we have consistently used the commercial [http://en.wikipedia.org/wiki/PUC19 pUC19-plasmid] (a high-copy plasmid containing ampicillin resistance). For the negative control we used competent cells undergoing the same procedure as the transformatants (minus the plasmid addition).

The transformation tubes didn't have any visible pellet after centrifugation in contrast to the electroporation tubes. Also the liquid containing the cells was less opaque for the transformation than the electroporation.

Day Two - GROWTH!

Growthcontrol. The transformation plates only showed growth on the negative control without ampicillin. However the electroporation plates showed growth on plates 2M, 6G, 18C and 23L. 18C showed very sparing growth and had a single colony, which we at the time interpreted as an contamination.

It later turned out that J61002 (which J23100=18C sits on) contains an RFP generator and didn't comply to Biobrick standard 10. So we scrapped 18C for a far better construct, namely 13B=J13002, a socalled expression plasmid containing a constitutive promoter(R0040) AND a RBS (B0034). . Also the insert in 3K (p1010=ccdB=Suicide gene) was active, which we hadn't realized prior to this point. Suicide genes properly explain no growth, so we switched to 1K=pSB1A3 with J04450(RFP generator).

Inoculation and streaking.

1 Colony is chosen from a growth plate and picked off with either a pipette-tip or a toothpick
Streak once through a new plate
Lower the pick into liquid medium and shake it all about
Using a new tip/pick expand the primary-streaking
Using a new tip/pick do secondary streaks
Using a new tip/pick do tertiary streaks

We prepared 23L, 6G and 2M each on three plates and three tubes and incubated them overnight at 37 degrees C. Tubes and plates both contain 100ug/mL Ampillicin

Special procedure for 18C. Because 18C had very little growth on it we made an attempt to save what was there:

Add 2mL of LB to the plate, distribute it evenly over the plate
Scrape of the LB and the colonies with it
Distribute 1mL in each of 2 eppendorf tubes
Spin at 14,5k RPM for 3minutes
Remove 900mL of supernatant by suction
100mL (incl. pellet) is plated onto 100ug/mL amp plates

We only got about 400mL off the plate again so be quick, we distributed it evenly among the tubes. For 5 it is more important that there is 100mL left than that you suck off 900mL of supernatant.

Day Three - Brick purification.

Using the growth from liquid medium we decided to grab us some concentrated plasmids. We achieved this through a process called miniprepping and controlled it via gel.

Miniprep. Using this protocol for miniprep we purified our plasmids. All centrifugation steps are performed at maximum speed (14,5k RPM).

Control by gel. To make sure that our miniprep had actually yielded the plasmids we were looking for we ran it through an 1,5% agarose gel. How to make an agarose gel. We loaded a mixture of 2uL miniprep-product, 1uL loading buffer and 2uL diH2O into wells in the gel along with an appropriate size marker. The bands on gel indicated that there was indeed size-appropriate chunks of DNA corresponding to our bricks in a plasmid backbone.

Day Four - Electroporation 1K & 13B.

Electroporation. When plating, we spilt a small amount of 13B2 due to the tube not being tightly sealed. We plated 3 plates for each of 1K and 13B. We also inoculated 3 LB tubes with 1K and 13B for miniprepping.

Day Five - Minipreppin'

Miniprep. As per protocol for miniprep.

Gel of miniprep-product. We ran

1uL Loading buffer

2uL diH2O and

2uL DNA from miniprep on a 1,5% (w/V) agarose gel along with an appropriate size marker. The sizes corresponded closely to the expected. Conclusion: We have bricks and backbone.

Week 3

Day One - Restriction day.

Protocol:

Pool plasmid and dry on vacuum centrifuge down to about 50uL
Mix the following into one tube:
1. 4uL Plasmid and RIP
2. 5uL 10x Buffer
3. 0,5uL BSA
4. Fill with water to 47uL (37,5uL)
5. 1,5uL Enzyme 1
6. 1,5uL Enzyme 2
Incubate for 2 hours on 37 degress C.
Inactivate the enzymes at 80 degress C for 20 minutes.
Place product on -20 degress C or continue immediately to ligation.

Restriction table

Name	Well	E	S	X	P
Inducible promoter	6G	X	X
RBS	2M			X	X
RIP		X	X
sRIP		X	X
Terminator	23L			X	X
Plasmid	1K	X			X
Constitutive promoter	13B	X			X

Notes on the restriction. RIP and sRIP pooled, vacuum dried till about 30uL, had to add a little water to RIP. Biobricks down to 50uL.

Gel of restrictions.

----PICTURE----

MORE CELLS. We're running low on competent cells, so we decided to make ourselves some more. Here is the protocol. Today we inoculated the MG1655 (the strain we are making competent)

Day Two - Charging forward.

Competent cells for electroporation. As per the protocol 2x200mL LB-medium was distributed in 2x4 50mL falcontubes og washed/spun/harvested. In the last round with water, a portion of the water was removed and the rest was pooled in 2x1 Falcontubes. The remaining pellet was dissolved in 1mL glycerol (deviation from protocol) and distributed in tubes with 40uL (mostly, we also did a few with 100uL). The tubes were INSTANTLY put in a rack placed in ethanol and dry ice, from which they were transferred to -80 degrees C.

2x40uL was taken out for controls. The positive control was electroporated with [http://en.wikipedia.org/wiki/PUC19 pUC19] and plated on 100ug/mL AMP LA plates. The negative control underwent the same treatment as the positive, only with no plasmid. Negative controls were plated on both 100ug/mL AMP LA plates and regular LA plates.

Growth result showed plenty of growth on the postive control. decent growth on negative without AMP and none on negative with AMP. Conclusion: The cells are definately competent.

Test Gel for Restrictions. Showed no sign of our bricks, plasmids were clearly visible, so were RIP and sRIP. We forgot to analyse by length of the bands and as such have no compelling proof for either succesful or unsuccesful restriction.

Miniprep PCR. We decided to do a PCR amplification step on our miniprep products, as it turned out that our PCR primers (VR and VF2) will work on all of our biobrick source plasmids. We use the same protocol as for Primer-PCR, except for point 3 in which we changed the temperature to 55 degrees C to fit with the new primers.

Restriction of backbone. We restricted some more backbone with EcoRI and PstI using the same Protocol as earlier.

Test Gel for PCR. We ran a test gel for our PCR-products. It's looking good. We'll pool and dry up the PCR-products, before purifying them via gel. After purification we will proceed to restriction. Under purification, we got a little over-eager and

Ligations. We attempted a ligation of:

Promoter + RBS
RIP + Terminator
sRIP + Terminator

Using this ligation protocol, since we didn't use backbone in this we dissolved to the same volume with 7uL H2O.

Day Three - Purifying backbone.

Freezetube preparation. In order to create a long term storage for our transformed strains we streaked 1K, 2M, 6G, 13B and 23L onto new 100ug/mL AMP plates.

Test Gel for backbone restriction. 2 out of 6 samples show 2 band. This is an indication that in some cases the plasmid has only been cut in one site. One-site restriction leaves us with 2 linear plasmids, of which 1 contains a biobrick (standard insert - RFP Generator - ). In order to purify our backbone we ran it all on a gel and excised only the bands corresponding to the properly cut backbone.

Purification of cut backbone from gel. We did 6 purifations in 9 tubes, here are the weights of each tube

	1	2	3	4	5	6	4b/7	6b/8	3b/9
Weight	0,20g	0,17g	310mg	290mg	0,27g	200mg	290mg	180mg	300mg

Week 4

Dear diary.

This week we worked hard on getting our ligations transformed into cells.

Day three

There was no growth in the backbone ligated with sRIP and RIP. All the controls worked, so the electroporation is working. So we still have problems with either our cuttings or ligations.

We decided to cut each biobrick again.

To test that our enzymes are working, we're going to cut each brick with both enzym 1 and enzym 2, and both enzym 1 and 2. Also we're testing both 30 min and 60 min cutting-time, so for each brick, we're going to have 6 samples. These we're going to run on a gel together with a non-cut brick. Hopefully we'll be able to see how well each biobrick is cut.

We made a new batch of PCR'ed BioBrick, so we have plenty to work with. Tomorrow we are going to run them on a gel, to get a more pure product and then cut that purified product.

After having looked at earlier gels, we concluded that there's isn't alot of DNA in our cut backbone (1K) and decided to pool it all in two eppendorf tube. Hopefully this will give us a better result. (mike)

Day four

Continuing on creating new biobricks from yesterday. Today we ran them on a gel, cut the band that corresponded to the right length, and then purified that gel-piece with a mini prep. After that, we ran 6 different cuts with each biobrick, 2 with the first enzym, 2 with the second enzym and 2 with both enzymes. We then ran one of each for 30 minutes, and another one of each for 1 hour. The idea is to run a small sample of all of them, to compare and check if our cuttings are working correctly. (mike)

Good news! It turns our we have growth on our plates from yesterday after all. We left them for another day at 37 degress celcius, and voila, now there are pretty colonies on the plates. Marc and Julius started the colony-pcr procedure.

Week 5

Day Two - Testing.

Dear diary, today we are beginning to get ahold of the last week and a halfs chaos.

Purification of PCR double-bricks

PCR product RIP-23L and sRIP-23L was mixed with 5uL loading buffer og purified from 1,5%(w/V) agarose gel.

Bands excised and dissolved:

RIP-23L 55mg

sRIP-23L 114mg

Purified PCR product are stored on -20 degress C.

Gel of yesterdays restrictions

	Content	Expected length	Band 1	Band 2	Band 3	Smear (+/-)
1	sRIP	174	~150bp			-
2	23L	129	>2kbp	~170bp		-
3	pSB1C3+J04450	>3kbp	>2kbp	~2kbp	~1,4 kbp	-
4	RIP					-
5	6G		>10kbp	>2kbp		+
6	2M		~10kbp	>2kbp		+
7	13B		>2kbp			-

1,2 and 3 look promising, the rest, not so much.

Ligations

1+2+3 → 11 = sRIP-23L-pSB1C3

4+2+3 → 12 = RIP-23L-pSB1C3

5+6+3 → 13 = 6G-2M-pSB1C3

7+3 → 14 = 13B-pSB1C3

Nothing really about the ligation process...

Ligation gel

	Wanted length	Band 1	Band 2	Band 3	Smear (+/-)
1	2299	~3,5kbp	~2,2kbp	~1,2kbp	+
2	2239	~4,0kbp	~2,2kbp	~1,2kbp	+
3	2147	~5kbp (very smeary)	~2,2kbp	~1,2 kbp	-
4	2146	~3,5kbp	2,2kbp	1,2kbp	-

Electroporation of ligationproducts

12 exploded :(

Purification of double-brick RIP-23L

This brick was expected at 313bp, but the closest visible band was at ~250-300bp (the other band was at <50bp). The area from the visible band and up to where the brick was expected was excsed and split equally into to tubes for purification. Stored overnight.

Team:SDU-Denmark/Diary