Team:Buenos Aires/ resqrfp

From 2013.igem.org

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= MINIPREP =
 
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==Materials==
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=mRFP under Arsenite Inducible Promoter ([http://parts.igem.org/Part:BBa_K1106003 Bba_K1106003]) characterization=
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* Resuspension buffer
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* Lysis buffer
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* Neutralization buffer
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* Isopropanol
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* 70% Etanol
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* dH2O
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* Ice (in ice bucket/container)
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==Procedure==
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'''Objective'''
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# Centrifuge for 5 minutes at 5000 rpm 1,5 ml of bacteria culture.
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Asess mRFP production, stability and naked eye discernibility range under inducible conditions.
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# Take the pellet and add 250 ul of resuspension buffer and pipet up and down a few times.(make sure that all is properly mixed)
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# Add 250 ul of Lysis buffer and invert the tubes 5 times to mix. Let rest 5 minutes. The mix must turn transparent.
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# Add 250 ul of cold Neutralization buffer and invert the tubes 5 times to mix. The mix must turn turbid.
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# Centrifuge at 13.000 rpm for 15 minutes.
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# Transfer the supernatant to a new clean eppendorf tube.
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# Add isopropanol (the same volume that is in the tube). Mix many times and put it in ice for 20 minutes.
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# Centrifuge at 13.000 rpm for 20 minutes.
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# Eliminate the supernatant (be carefull that the pellet does not go with it)
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# Add 1 ml of etanol 70% and use a vortex to ensure the pellet mixes with the etanol.
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# Centrifuge at 13.000 rpm for 5 minutes.
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# Eliminate all the supernatant with a pipette (be carefull that the pellet does not go with it)
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# Let the tube open until it is dried. The pellet must turn from white to transparent.
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# Add 20 ul of dH2O and freeze.
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 +
'''General procedure'''
 +
Different assays were performed using ''E. coli'' (DH5α strain) harbouring a plasmid that encodes mRFP under arsenite inducible promoter (ArsRFP culture).
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=Transformation=
 
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We have E. Coli DH5α strain competent bacteria, with a transformation efficiency of 10
 
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'''Important!''' Competent bacteria should always be on ice.
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== '''mRFP production at different arsenite concentrations''' ==
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# If the plasmid comes from the kit or is a product of ligation, add 2μl of plasmidic DNA into 50μl
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'''Method'''
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of competent bacteria. If it’s a plasmid that comes from a miniprep, use a mass in function of
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ArsRFP cultures were grown with different arsenite concentrations ( 0, 25, 50, 100, 500 and 1000ppb). 1ml aliquots were taken after 24 hours and fluorescence was measured.
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the efficiency of the bacteria.
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'''Results'''
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# Incubate for 20 min on ice.
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mRFP fluorescence increases with higher arsenite concentrations, in a sigmoidal way.
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# Heat Shock: 42°C for exactly 1’30’’, then immediately place on ice.
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[[File:Exp_Ine.jpg|center|600px|]]
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# Incubate for 5 min on ice.
 
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# Recovery: Add 950μl of LB without antibiotic, incubate from 30 min to an hour at 37°C
 
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# Pellet the bacteria by centrigue at 10000rpm for 5 minutes. Discard the supernatant of LB.
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<html>
 +
<br>
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<br>
 +
</html>
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Resuspend the bacteria in the remnant LB.
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==Measurement of groundwater samples with pars_mRFP==
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# Plaque onto plates with LB and the desired antibiotic.
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Using the experiment above as a calibration curve, we calculated the concentration of arsenite in groundwater samples. To achieve that, we first had to linearize the curve. As the curve seems to fit a logarithmic function, we aplied a logarithmic transformation to the original data, setting 1,6 as the base and the value of fluorescence at 608 nm/OD as the exponent. This way, we got a good linear fit, and used the function obtained to estimate arsenite concentration from values of fluorescence at 608 nm/OD.
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# Incubate overnight in a stove at 37°C
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[[File:Ajuste_lineal_RFP.png|center|600px|]]
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=Ligation=
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We then compared our method to an commonly used method for arsenic detection (Atomic Absorption Spectrometry, AAS). the results show that, despite pArs_mRFP construct is not as precise as AAS, it can be used as a method to indicate the arsenic concentration at different ranges (and that is what we actually need!).
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'''Important!''' Before starting the ligation process, make sure that the restriction enzymes are inactive.
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[[File:Correlacion_arsenico.png|center|600px|]]
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# Add 2μl of digested plasmid backbone (or the equivalent of 25ng).
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<html>
 +
<div id="inwiki">
 +
</html>
 +
For more information about the sample locations and their arsenic concentrations, please visit our [https://2013.igem.org/Team:Buenos_Aires/_mapaarsenico Human Practice section ]
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# Add equimolar amount of EcoR1-HF Spe1 digested fragment (less than 3μl).
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<html>
 +
</div>
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</html>
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# Add equimolar amount of Xba1 Pst1 digested fragment (less than 3μl).
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== '''mRFP production over time''' ==
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# Add 2μl of T4 DNA ligase buffer (final concentration should be 1x).
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'''Method'''
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# Add 0.5μl of T4 DNA ligase.
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A 100 ml ArsRFP culture was grown at 30°C until it reached OD=0.4 (OD 600nm). At this point arsenite was added (1000ppb final concentration) and fluorescence was measured every 30 minutes during 8 hours at 584 nm excitation peak and 608 nm emission peak.  
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# Add water to 10μl.
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'''Results'''
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# Ligate at 16°C for 30 min, then heat kill 80°C for 20min.
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As shown in the figure below, mRFP production increses over time with arsenite (1000 ppb). However, there is a 3 hours lag after inoculation.
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# Transform with 1 – 2 μl of product.
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[[File:RFP_induccion.jpg|center|600px|]]
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<html>
 +
<br>
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<br>
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</html>
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=LB & LB agar=
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== '''mRFP stability over time''' ==
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# Liquid LB (1 litre)
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'''Method'''
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# 10g of bacto peptone (peptone, triptone or bactereologic triptone)
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ArsRFP culture was grown overnight at 37ºC in 10 ml LB medium with 2000ppb arsenite concentration. The following day, the culture was centrifuged and the supernatant was discarded in order to remove the arsenite, thus stopping the induction. Afterwars, fresh LB medium was added and the pellet was resuspended. This was done twice and the culture was returned to 37ºC incubation. 1 ml of this culture was taken every 12 hours for the following 4 days. Finally, fluorescence was measured.
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# 5g of yeast extract
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'''Results'''
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# 10g of NaCl
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mRFP degradation is shown over time, specially during the first 24 hours.
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# Fill with dH2O
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[[File:RFP_santi.jpg|center|600px]]
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Autoclave.
 
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TIP: if it is not going to be autoclaved in the next few hours water should not be added (because even
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<html>
 +
<br>
 +
<br>
 +
</html>
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when in the fridge it will get contaminated), in which case everything can be measured and left ready to
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== '''Naked eye discernibility range of mRFP production by arsenite inducible promoter''' ==
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just add the water.
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'''Method'''
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# LB agar (1 litre)
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ArsRFP cultures were grown with different arsenite concentrations ( 0, 10, 50, 200 and 1000ppb). 1ml aliquots were taken every 12 hours and centrifuged at 10.000rpm for 5 minutes. Pellets pictures were taken in order to compare the range of colour at naked eye. mRFP fluorescence was also measured with a fluorimeter at 484nm for excitation and 608 nm for emission.
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# 10g of bacto peptone (peptone, triptone or bactereologic triptone)
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'''Results'''
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# 5g of yeast extract
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As it can be seen in the pictures below, a difference in the colour production can be clearly distinguished between different arsenite concentrations after 24 hours of induction and between the higher arsenite concentrations only. It can also be observed that over time the production grows and that after 62 hours the difference between 200 and 1000ppb is not clear.
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# 10g of NaCl
 
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# 15g of agar
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{| class="wikitable" style="background-color:#fff;margin:auto;"
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|+Induction over time at different concentrations of arsenite (ppb)
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|-
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|After 12 hours of induction:
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[[File:T1rfp.jpg | center | 400px]]
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|-
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|After 24 hours of induction:
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[[File:T2rfp2.jpg| center | 400px]]
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|-
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|After 36 hours of induction:
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[[File:T3rfp.jpg | center | 400px]]
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|-
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|After 48 hours of induction:
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[[File:T4'rfp.jpg | center | 400px]]
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|-
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|After 50 hours of induction:
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[[File:T5rfp.jpg | center | 400px]]
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|-
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|After 62 hours of induction:
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[[File:T6rfp.jpg| center | 400px]]
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|}
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# Fill with dH2O
 
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'''IMPORTANT''': agar does not dissolve if not heated so if Erlen-Meyers are prepared with LB agar, add LB in
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== '''Collaboration with iGEM Tec-Monterrey team''' ==
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liquid form and then add the agar to the Erlen-Meyer according to the volume.
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We established a collaboration with the iGEM Tec-Monterrey team, and exchanged constructions to characterize. In this link you may find their characterization of our mRFP under pArs: [[https://2013.igem.org/Team:TecMonterrey/collab_argentina.html Tec-Monterrey characterization]].
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Autoclave.
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'''TIP''': if it is not going to be autoclaved in the next few hours water should not be added (because even
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+
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when in the fridge it will get contaminated), in which case everything can be measured and left ready to  
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just add the water.
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=Gel=
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TAE 1x (diludde from TAE stock solution – 50x)
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Agarose
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Remember to use gloves during the entire process, being mindful not to have direct contact with
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ethidium bromide.
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# Add the necessary agarose into an Erlenmeyer flask and then add TAE, and fill with water. The
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final concentration of agarose may depend on the type of gel needed, but usually will be 1%.
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#  Heat the flask, while covered,  in a microwave until completely dissolved (should take no more
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than 3 minutes at maximum potency)
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#  Add the necessary volume of Ethidium Bromide (about 10 μl for each 100g of gel)
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'''IMPORTANT''': Ethidium Bromide is carcinogenic. It should be handled with gloves, and these,
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such as anything that has come in direct contact with Ethidium Bromide must be discarded
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separately in an assigned discard bag.
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# Pour the melted gel, with Ethidium Bromide, slowly into the gel tray. Add the combs and leave
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until solified. Remove the combs. Rinse the flask with abundant water.
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#  Place the gel tray inside the gel box being mindful of the direction in which gel will run. Add TAE
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1x until it is completely covered.
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 +
== '''Overall conclusions''' ==
 +
mRFP production responds efficiently under inducible conditions, both over time and different arsenite concentrations. The mRFP stability is acceptable for the aim of our Project. However, the visibility to the naked eye is not sufficient at low arsenite concentrations.
 +
We reached to the conclusion that, in order to increase the colour production at lower arsenite concentrations, a signal amplification system has to be added. It could also be added some switch to stop the production and avoid saturation of all the samples, and thus keep the range of colour observable to the naked eye.
</div>
</div>

Latest revision as of 14:24, 28 October 2013

Contents

mRFP under Arsenite Inducible Promoter ([http://parts.igem.org/Part:BBa_K1106003 Bba_K1106003]) characterization

Objective

Asess mRFP production, stability and naked eye discernibility range under inducible conditions.

General procedure

Different assays were performed using E. coli (DH5α strain) harbouring a plasmid that encodes mRFP under arsenite inducible promoter (ArsRFP culture).


mRFP production at different arsenite concentrations

Method

ArsRFP cultures were grown with different arsenite concentrations ( 0, 25, 50, 100, 500 and 1000ppb). 1ml aliquots were taken after 24 hours and fluorescence was measured.

Results

mRFP fluorescence increases with higher arsenite concentrations, in a sigmoidal way.

Exp Ine.jpg




Measurement of groundwater samples with pars_mRFP

Using the experiment above as a calibration curve, we calculated the concentration of arsenite in groundwater samples. To achieve that, we first had to linearize the curve. As the curve seems to fit a logarithmic function, we aplied a logarithmic transformation to the original data, setting 1,6 as the base and the value of fluorescence at 608 nm/OD as the exponent. This way, we got a good linear fit, and used the function obtained to estimate arsenite concentration from values of fluorescence at 608 nm/OD.

Ajuste lineal RFP.png

We then compared our method to an commonly used method for arsenic detection (Atomic Absorption Spectrometry, AAS). the results show that, despite pArs_mRFP construct is not as precise as AAS, it can be used as a method to indicate the arsenic concentration at different ranges (and that is what we actually need!).

Correlacion arsenico.png

For more information about the sample locations and their arsenic concentrations, please visit our Human Practice section

mRFP production over time

Method

A 100 ml ArsRFP culture was grown at 30°C until it reached OD=0.4 (OD 600nm). At this point arsenite was added (1000ppb final concentration) and fluorescence was measured every 30 minutes during 8 hours at 584 nm excitation peak and 608 nm emission peak.

Results

As shown in the figure below, mRFP production increses over time with arsenite (1000 ppb). However, there is a 3 hours lag after inoculation.

RFP induccion.jpg



mRFP stability over time

Method

ArsRFP culture was grown overnight at 37ºC in 10 ml LB medium with 2000ppb arsenite concentration. The following day, the culture was centrifuged and the supernatant was discarded in order to remove the arsenite, thus stopping the induction. Afterwars, fresh LB medium was added and the pellet was resuspended. This was done twice and the culture was returned to 37ºC incubation. 1 ml of this culture was taken every 12 hours for the following 4 days. Finally, fluorescence was measured.

Results

mRFP degradation is shown over time, specially during the first 24 hours.

RFP santi.jpg




Naked eye discernibility range of mRFP production by arsenite inducible promoter

Method

ArsRFP cultures were grown with different arsenite concentrations ( 0, 10, 50, 200 and 1000ppb). 1ml aliquots were taken every 12 hours and centrifuged at 10.000rpm for 5 minutes. Pellets pictures were taken in order to compare the range of colour at naked eye. mRFP fluorescence was also measured with a fluorimeter at 484nm for excitation and 608 nm for emission.

Results

As it can be seen in the pictures below, a difference in the colour production can be clearly distinguished between different arsenite concentrations after 24 hours of induction and between the higher arsenite concentrations only. It can also be observed that over time the production grows and that after 62 hours the difference between 200 and 1000ppb is not clear.


Induction over time at different concentrations of arsenite (ppb)
After 12 hours of induction:
T1rfp.jpg
After 24 hours of induction:
T2rfp2.jpg
After 36 hours of induction:
T3rfp.jpg
After 48 hours of induction:
T4'rfp.jpg
After 50 hours of induction:
T5rfp.jpg
After 62 hours of induction:
T6rfp.jpg


Collaboration with iGEM Tec-Monterrey team

We established a collaboration with the iGEM Tec-Monterrey team, and exchanged constructions to characterize. In this link you may find their characterization of our mRFP under pArs: [Tec-Monterrey characterization].

Overall conclusions

mRFP production responds efficiently under inducible conditions, both over time and different arsenite concentrations. The mRFP stability is acceptable for the aim of our Project. However, the visibility to the naked eye is not sufficient at low arsenite concentrations. We reached to the conclusion that, in order to increase the colour production at lower arsenite concentrations, a signal amplification system has to be added. It could also be added some switch to stop the production and avoid saturation of all the samples, and thus keep the range of colour observable to the naked eye.

Retrieved from "http://2013.igem.org/Team:Buenos_Aires/_resqrfp"