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From 2009.igem.org

Laura's lab journal

Goal and objectives

At the start I was working with the delivery system which includes P4 and P2 bacteriophages. However, on August I focused my attention on the phages involved in the defense system, T7 and T3.
My main goals were:

• To obtain all the relevant experimental information about T7 and T3 as the burst sizes and the growth plots with and without phage.
  
• To generate data to feedback the infection model.
  
  

I used two main protocols the Purification of T7 from 35ml lysates from the T7Select System Manual and the Titering Protocol described in the same Manual.

The activities ordered chronologically are described next.

August

On August I was working on verifying the accuracy of these protocols and on select a strain for the following experiments. In this month a purification protocol was conducted on Escherichia coli K-12 strain, the lysis was unsuccessful. To probe the activity of the phage stocks I got the BL21 strain to work with T3 and T7, a purification protocol was conducted on Escherichia coli BL21 strain, the lysis was successful. T3 and T7 bacteriophages stocks were active. It was decided to use C1a to work with T3 and T7 because it is a K-12 derivative. In the following experiments were be focused on this strain. The purification of T7 and T3 using C1a strain was successful (OR stock)
In this month all the cultures got contaminated with yeast (strains C331, C-117, C1a, C-1906, c520, c1895, c2423). The contaminated cultures were transferred to generate spectomycin resistant. Resistant strains c-117, c1a, c331 and c1906 were obtained. I did PCR’s to be sure about the genome integrity. I amplified Cox for c117, c1a and c331; Ogr for c117, c331 and c1a and P4 for c1906, c1a and c331. I charged an agarose gel to check the PCRs, cox and ogr were seen when they were supposed to be. The resistant strains were contaminated with yeast. So, I did no more efforts on this. New stock of the main C1a stock was obtained.

September

From a culture with OD=0.8 I transferred 1.25ml to have a 100ml culture with OD=0.01. Every four hours for a period of 12 hours I took 1ml from this culture to take an OD measure(to 550nm) and I took another ml to do dilutions, I did cultures corresponding to the 10^-4, 10^-5 and 10^-6 dilutions, and an additional 10^-3 dilution for the first measure. I counted the number of UFCs (unit forming colonies) for these cultures and I calculated the number of UFC per ml in the original culture at time in which the sample was taken. After this, I used a non linear regresion method to obtain the logistic formula with the best adjustment to the points for each plot.The corresponding plots and formula are presented next.

 tiempo (hrs)	DO 550nm	UFC/ml
 0		0.0355		1207000
 2		0.2533		25450000
 4		0.944		160000000
 6		1.2194		165000000

Both formulas were used to infer a correlation formula between OD and UFC.

The purification protocol was repeated three times for each phage and every stock (1BS, 2BS and 3BS) was tittered including the OR stock. I always followed the protocol of T7 from 35ml lysates from the T7 Select System Manual protocol and the tittering protocol from the same manual. Here I present some important details about these experiments.

For the first purification (1BS stock) I mixed 5ul of T7 OR stock with 34ml of M9LB plus c1a(DO 0.84) and 5ul of T3 OR stock with 33ml of M9LB plus c1a (OD 0.85). It was left by three hours to reach a total lysis. For the tittering of this stock I did dilutions, I took 100ul of the dilution and mixed it with 350ml of cells and with 3ml of top agar. In the next purification procedures I only change the volume and the OD of the culture. For the second purification it was 32ml with 0.77OD for both, T7 and T3. For the third purification it was 34ml for both bacteriophages with a OD=0.94 for T3 and OD=0.94 for T7. The tittering for each stock was repeated some times, the results are presented in the following tables. The non congruent experiments between the dilutions are not presented here.

The number of plaques obtained for each experiment are shown in the following tables (the numbers after the name of the stock indicate the number of the replicate)

T7

  DILUTION	OR1	OR2	OR3
  10^-8		167	76	50
  10^-9		16	7	5
  10^-10	2	0	0

 DILUTION	1BS1	1BS2	2BS1	2BS2	3BS1	3BS2	3BS3
 10^-9		417	210	NC	NC	Clear	Clear	Clear
 10^-10		37	25	NC	NC	Clear	Clear	Clear
 10^-11		4	3	480	480	NC	Clear	Clear
 10^-12		---	---	----	---	364	NC	NC

NC=non-countable, Clear = all the cells were dead, ---=the experiment wasn't done.

T3

 DILUTION	OR1	OR2	1BS1	1BS2	1BS3	IBS4	2BS1	2BS2	2BS3
 10^-8		350	104	NC	Clear	Clear	Clear	NC	NC	Clear
 10^-9		30	10	210	Clear	NC	Clear	480	182	NC
 10^-10		3	0	25	NC	254	NC	63	23	600
 10^-11		---	---	3	250	25	251	8	2	74

The same nomenclature was used

The results were conclusive for:

*T7 OR stock: 1x10^8 UFP/ul
*T7 1BS: 3.72x10^12 UFP totales
*T7 2BS:5.76x10^14 UFP totales
*T7 3BS: one or two magnitude order above the last result (the data are not enough to specify a number).
*T3 OR: 2x10^8

Burst size obtained with purification data.

These results were used to obtain the “burst size” of T7 bacteriophages. I assumed a couple of things: all the phages are infecting a bacterium, all the cells were infected and almost all the bacteria died in the first round of infection. I calculated the bacteria number with the formula obtained from the growth curve which involves OD and UFC. So I calculated the burst size as the total number of phages produced divided by the total number of bacteria. The data from the first purification (1BS stock)indicate a burst size of 952 phages per bacterium. However the data from the 2BS stock indicate a burst size of 160,000 phages per bacterium and surprisingly the data from the 3BS stock indicate a bigger number. We know it is not possible because the phage production is limited by the amount of nucleotides available. For this reason, We designed other experiment to measure the burst size. From now I focused on T7 because I felt the time wasn't enough.

October

We did a growth curve with phage.

In the first assay I cultured 35ml of M9LB c1a when the OD reached a value close to 0.8 I added 5ul of T3 or T7. I took OD measures (550nm)every twenty minutes. No phage was added in the control. The data recollected from this experiment is shown in the following table. I did the assay twice. Because I took different initial values for this replicate and because I didn’t do plates I repeated the assay taking into account these facts.

  Time(min)	T7(1)(OD)	T7(2) (OD)	T3(1) (OD)	T3(2) (OD)	C1(OD)	C2(OD)
  0(0)		0.69		0.83		0.71		0.72		0.65	0.79
  1(20)		1.02		1.15		1.04		1.05		0.96	1.13
  2(40)		0.45		0.61		0.52		0.52		1.32	1.39
  3(60)		0.24		0.25		0.23		0.25		1.52	1.59
  4(80)		0.24		0.25		0.23		0.25		1.66	1.70

In the second assay the same procedure was used, but this time I did plates on time 0 (dilutions 10^-4 and 10^-5) and on time 5 (dilutions 10^-3 and 10^-5) and I took the same OD initial values for both replicates, the mean values are presented next and are represented in the plot.

 Time(min)	T3(OD)	T7(OD)	C(OD)
 0(0)		0.78	0.78	0.8
 1(20)		1.02	1.02	1.06
 2(40)		0.57	0.58	1.27
 3(60)		0.305	0.41	1.47
 4(80)		0.29	0.275	1.59
 5(100)		0.29	0.279	1.61

When I went over the plates I realized the results for the time 0 were inconsistent, however on time 5 plates I obtained 2 colonies for the 10^-3 T3 and T7 plates, 0 colonies for the 10^-5 T3 and T7 plates, for the control plates I almost got a bacteria monolayer in 10^-3 plate and non-countable bacteria in 10^-5.

Second experiment to obtain the burst size.

I did two replicates of this experiment. I did two 25ml cultures. When the culture had an OD around 0.8 (0.738 and 0.724, respectively)I put 100ul of some phage dilutions. I supposedly added seven and five phages, respectively. I waited for 20 minutes, it is supposed that the lysis lasts approximately 14 minutes. After this I follow the purification protocol adding 3ml of NaCl and 4ml of PEG 6000% in the right time. In the next step I did phage dilutions (10^-1, 10^-2 and 10^-3) and I followed the Titering Protocol. No one plaque was observed after three hours. I suppose the Purification Protocol was unsuccessful because the small amount of phages.

Third experiment to obtain the burst size.

I did two replicates of this experiment. I did a modified Tittering Protocol. I took 500ul of cells with an OD of 0.8 and I added 100ul of phage dilution. The dilutions were made from the T7 original stock, I used the 10^-8, 10^9, 10^10 and 10^11 dilutions in which I expected 100, 10, 1 and 0 phages, respectively. This mix stayed 25 minutes on the shaker after this I tittered the cultures and I obtained the next results.

 DILUTION	3E		3E2
 10^-8		Clear		Clear
 10^-9		Clear		Clear
 10^-10		1000 aprox	1000 aprox
 10^-11		1000 aprox	1000aprox

The same nomenclature rules are followed

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