Team:UTP-Panama/Experiments4

From 2011.igem.org

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== BCCS-Bristol 2010 Biobrick ==
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== New Experiments ==
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===1. First Experience with BBa_k381001===
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===Further Characterization===
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Objectives:
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1. Perform further experiments with our device
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2. Improve the characterization of THE RENBO
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3. Determine the relationship between the parameters we are studying (gene expression, temperature, substrate and thermal method)
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4. Obtain the proper functioning of our device
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Methodology:
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 We will use 5mL of substrates (liquid LB and MME)
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 We will have controls with and without antibiotics
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 We will use colonies of the following:
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o JM109 (just the strain with empty plasmids, control)
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o With RFP (control)
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o Bristol BBa_K381001
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o GaTech  BBa_K672000
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 Temperatures we used are:
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o 37ºC
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o Between 15ºC and 20ºC  (18ºC)
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 We will study growth using the two thermal methods:
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o Studying growth putting the colonies to grow directly at the chosen temperatures
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o Studying growth when the colonies have undergone a thermal shock @10ºC for 1 hour
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 For each temperature and BioBrick we wanted to get (in each experiment):
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o Growth curve
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o measurement of GFP expression
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o measurement of RFP expression (control)
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o measurement of heat production (thermometer)
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<div align="center">Experiments:</div>
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Objective:<br>
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#1: All Colonies in LB and MM in blank, for references without antibiotics and without nitrate, @37ºC and @18ºC.
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1. Test operation in liquid LB at 37 ° C and to KNO3 50 mM.<br>
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#2: All Colonies in LB and MM with nitrate @37ºC and @18ºC.
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#3: All Colonies in LB and MM with antibiotics @37ºC and @18ºC.
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Methodology:<br>
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#4: All Colonies in LB and MM with antibiotics and nitrate @18°C and @37°C, first passing through thermal shock @10°C for 1 hour.
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#5: All Colonies in LB and MM with antibiotics and nitrate @18°C and @37°C, without thermal shock, direct growing.
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1. We used six falcon tubes of 25 mL. Three tubes were used for inoculation of the colonies from 06/09/11 and 12/09/11.<br>
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2. Two positive controls were prepared and one negative control for each test.<br>
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3. To positive control tubes we added 10 ml of liquid LB, 9.7 uL of chloramphenicol (33μg/mL) and 50mM of KNO3.<br>
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4. To negative control tube we added the same amount of LB and Chloramphenicol specified in the previous line, except KNO3.
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5. Each tube was inoculated with the colonies; three of them with the conlonies from 06/09/11 and the rest with colonies from 12/09/11.<br>
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6. Tubes were incubated at 37 ° C for 48 hours.<br>
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After 48 hours we noticed that all the tubes for both tests showed no
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change, including positive and negative controls. Our experiment was
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based on BCCS-Bristol experiment. While reviewing the experiments of
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Edinburgh in 2009, who designed the promoter used in the BioBrick
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BCCS-Bristol, we realized that the promoter express better in minimal
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essential media than in LB, but we do not rule out that we could have
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made a mistake in the experience preparation prevented us from
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showing the right results, which is why we decided to do the experiment
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again.
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===2. Second Experience===
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Objectives:<br>
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1. Test the BioBrick operation to different substrates and temperatures, using KNO3  40 mM:<br>
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a. Substrates: <br>
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*LB <br>
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*Minimal essential medium<br>
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*Saline solution<br>
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b. Temperatures:<br>
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*8°C<br>
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*23°C<br>
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*37°C<br>
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2. Improve the characterization of the BioBrick tested in other substrates to study the performance of the promoter.<br>
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3. Define the Biobrick promoter strength at the temperatures and selected substrates.<br>
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'''Methodology'''<br>
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According to the characterization made by Team Edinburgh 2009, the Pyear promoter (BBa_K216005) used by Bristol 2010, works
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best in minimal media and complex media with a nitrate concentration of 40mM. Using this reference, we added 40mM of KNO3 to
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every growth media in the different temperatures for Bristol 2010 - BBa_K381001 and UTP-Panama 2011 – BBa_K672000.<br>
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4. We used nine falcon tubes of 15 mL. Three tubes for every different substrate at the three temperatures.<br>
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5. To each tube we added 10 ml of liquid LB, Minimal Media or Saline Solution and KNO3 40 mM.<br>
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6. Tubes corresponding the different substrates were inoculated with BBa_K381001.<br>
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7. Tubes were incubated at 37°C, 8°C and 23°C for 24 hours.<br>
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Latest revision as of 03:57, 29 October 2011

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New Experiments

Further Characterization

Objectives: 1. Perform further experiments with our device 2. Improve the characterization of THE RENBO 3. Determine the relationship between the parameters we are studying (gene expression, temperature, substrate and thermal method) 4. Obtain the proper functioning of our device Methodology:  We will use 5mL of substrates (liquid LB and MME)  We will have controls with and without antibiotics  We will use colonies of the following: o JM109 (just the strain with empty plasmids, control) o With RFP (control) o Bristol BBa_K381001 o GaTech BBa_K672000  Temperatures we used are: o 37ºC o Between 15ºC and 20ºC (18ºC)  We will study growth using the two thermal methods: o Studying growth putting the colonies to grow directly at the chosen temperatures o Studying growth when the colonies have undergone a thermal shock @10ºC for 1 hour  For each temperature and BioBrick we wanted to get (in each experiment): o Growth curve o measurement of GFP expression o measurement of RFP expression (control) o measurement of heat production (thermometer)

Experiments:
  1. 1: All Colonies in LB and MM in blank, for references without antibiotics and without nitrate, @37ºC and @18ºC.
  2. 2: All Colonies in LB and MM with nitrate @37ºC and @18ºC.
  3. 3: All Colonies in LB and MM with antibiotics @37ºC and @18ºC.
  4. 4: All Colonies in LB and MM with antibiotics and nitrate @18°C and @37°C, first passing through thermal shock @10°C for 1 hour.
  5. 5: All Colonies in LB and MM with antibiotics and nitrate @18°C and @37°C, without thermal shock, direct growing.
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