Team:University College London/LabBook/Week11

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== (11.1) Monday 20.08 ==


'''Aim:'''  To test how the amount of nuclease produced by W3110 E. coli cells changes over time (and varies with the size of the colony?). 


'''Methods:''' 
For Wnu cell line which has native secreted nuclease activity
1.	Prepare 11-16 plates (10ml LBAgar+10ul AMP +10 ul 1M IPTG). IPTGinduces the lac promoter which in turnactivates the transcription of nuclease.
2.	Streak cells onto all plates at the same time
3.	Incubate at 37°C 
4.	Applyhydrchloric acid (HCL) to the first plate before putting in the incubator (set time as zero)
5.	Take a second reading after four hours, followed by six readingsevery 3 hours, and a final  three readings every two hours.
6.	When the reading is taken, observe the following:
a)	Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)
b)	Diameter of the halo that is achieved once HCL is applied 
c)	OD from a)
d)	Estimate of the depth of the colony on the agar plate
For BL21 cell line that has been modified to contain nuclease
7.	Prepare 11-16 plates (LB Agar + CMP)
8.	Streak cells onto all plates at the same time
9.	Incubate at 37°C 
10.	Apply HCL to the first plate before putting in the incubator (set time as zero)
11.	Take a second reading after four hours, followed by six readings every 3 hours, and a final  three readings every two hours. 
12.	When the reading is taken, observe the following:
a.	Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)
b.	Diameter of the halo that is achieved once HCL is applied 
c.	OD from a)
d.	Estimate of the depth of the colony on the agar plate


'''Results:''' The following table shows readings of the colony and halo diameters, plus the OD, taken at different times over 28 hours. 

Date	Time	Colony diameter	Halo diameter	Absorbance at 600 OD
20.08.2012	12.30pm	0	0	0
20.08.2012	16.30pm	0	0	0
20.08.2012	19.30 pm	0	0	0
20.08.2012	22.30 pm	0.5mm	1mm	0.001
21.08.2012	01.30 am	1mm	3mm	0.142
21.08.2012	04.30 am	1.5mm	7mm	0.228
21.08.2012	07.30 am	1.5mm	7.5mm	0.252
21.08.2012	10.30am	2.5mm	11mm	0.522
21.08.2012)	12.30pm	3mm	13mm	0.749
21.08.2012	14.30pm	3.5mm	14.5mm	0.844
21.08.2012	16.30pm	4mm	16mm	0.937


'''Conclusion:''' From the data collected, it is clear that colony size correlates with the nuclease produced, which is represented by the diameter of the halo diameter. Both are proportionally related to time. The second run of the experiment will be done in a period of several weeks so that we would have data replicates in order to validate data (See Week 13).

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== 11.2 ==
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== 11.3 ==
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== 11.4 ==
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