Difference between revisions of "Part:BBa R0011:Experience"

Latest revision as of 03:14, 18 October 2018

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_R0011

User Reviews

UNIQfe25387222dc9d52-partinfo-00000000-QINU

•••••

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

UNIPV-Pavia iGEM 2009

UNIPV-Pavia iGEM 2009's Experience: BBa_R0011 - Plac hybrid promoter

•••

UNIPV-Pavia iGEM 2010

UNIPV-Pavia iGEM 2010's Experience

Washington 2010

Washington 2010's Experience: R0011 in different plasmids

••••

iGEM Kyoto 2010

We measured RPUs of R0011 in low number copy plasmid with various concentrations of IPTG

See details.

••••

iGEM11_Uppsala-Sweden

We measured RPUs of different promoters in low copy number plasmids.

See details.

;

•••••

UT-Tokyo 2011

UT-Tokyo 2011 Team has characterized this part through the test of our Firefly-Renilla Dual Luciferase Assay Kit. We succesfully evaluated the relative expression levels of BBa_R0011 with various IPTG concentration, compared to that of BBa_J23119 as a control. For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].

;

•••••

[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012]

iGEM Team Uppsala University 2012

Promoter strength

Promoter strenghts in MG1566 and DH5alpha, relative to J23101.

A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (BBa_K864101) in the backbone BBa_K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.

Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.

	MG1566	DH5alpha
J23101	1	1
J23106	0.19	0.37
J23110	0.27	0.50
J23116	N/A	0.11
Plac	0.34	0.67
PlacIq	0.03	0.05
T5lac	0.217	0.54
PLlacO	0.87	N/A

The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.

LacI repression

The possibility of repression by lacI and induction by IPTG was evaluated in the pSB4C15Iq backbone. Cells grown with IPTG (0.5 mM) had a 100-fold increase of RFP expression, when compared to those grown without. Read about pSB4C15Iq for details.

•••••

[http://2013.igem.org/Team:UCSF UCSF 2013]

The UCSF iGEM team tested the pLAC promoter (part BBa_R0011) by placing it in front of GFP and inducing with various concentrations of IPTG. We used this part in the design of our CRISPRi circuit and improved upon it through the following experiments:

Time curve for original pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader over time. GFP fluorescence were corrected for OD600 value. Error bars indicate standard deviation calculated on the basis of technical replicates.
Dose-response curve for original pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.

•••••

[http://2018.igem.org/Team:NDC-HighRiverAB NDC-HighRiverAB]

QUALITATIVE DATA

We first did a qualitative test. From the literature, we found that we could use 4-nitrophenyl palmitate and 4-nitrophenyl octanoate to test our esterase activity. Both substrates, when cleaved by an esterase, would go from clear to green, allowing us to monitor the cleavage activity visually. We set-up a time-course assay where we mixed 1mM of each nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer. We also tested DH5⍺ cells without any plasmid as well as the ester mixture with no cells as controls. Figure 2 Shows the results from this experiment.

From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.

We did not add IPTG to these tests, so the green colour indicates that our promoter was "leaky", allowing continual expression of our EstA without the need of IPTG.

A comparison of 4-nitrophenyl palmitate and 4-nitrophenyl octanoate

Figure 2: The top row shows centrifuge tubes containing the 4-nitrophenol ester reaction mixture for the palmitate (a) and octanoate (b) esters with no cells added. The middle row shows the response for a negative control where DH5⍺ cells without any plasmid were added to reaction mixtures containing 4-nitrophenyl-palmitate (c) or 4-nitrophenyl-octanoate (d). The bottom row shows the response for the BBa_K2694000 circuit transformed into DH5⍺ cells when added to reaction mixtures containing 4-nitrophenyl-palmitate (e) or 4-nitrophenyl-octanoate (f). All reaction mixtures were 1mM of the nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer.

From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.

QUANTITATIVE DATA

Once we had shown that our bacteria could cleave the substrates to some extent, we wanted to try to show this more quantitatively. In collaboration with the University of Calgary’s iGEM 2018 team, we designed an experiment to test our reaction in a time course, using their spectrophotometer, in order to more accurately determine the color change.

Graph 1 shows the averages of the results from all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl octanoate with and without IPTG. Our control started at 0 and ended at 0.002. All other samples increased for about 660 seconds and then started to level out. This graph shows that our part, BBa_k2694000 in E.coli was producing the EstA protein because the solution got progressively more green with the introduction of our bacteria, but without the bacteria the control did not change colour. Based on these averages, the IPTG did not appear to have an effect on our reaction under these conditions. Our bacteria passively produces the repressor for pLac, but since we added more pLac to the system, there may not have been enough of the repressor to inhibit the production of our EstA, which may explain why IPTG did not have any effect on the reaction.

Graph 1: Average Absorbance Rates for the Breakdown of 4-Nitrophenyl Octanoate using BBa_K2694000 With and Without IPTG.

Graph 2 shows the averages of the results from all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl palmitate with and without IPTG. Our control started at 0 and ended at 0.002. All other samples did not increase significantly. This shows that BBa_K2694000 is not good at breaking down the 4-nitrophenyl palmitate under these conditions. This further shows that IPTG did not have a noticeable effect on the expression of our gene.

Graph 2: Average Absorbance Values for the Breakdown of 4-Nitrophenyl Palmitate by BBa_K2694000 With and Without IPTG.

UNIQfe25387222dc9d52-partinfo-0000000D-QINU

@@ Line 5: / Line 5: @@
 ===Applications of BBa_R0011===
-====BBa_R0011 - Plac hybrid promoter - UNIPV-Pavia team (test performed by L. Pasotti, S. Zucca)====
-=====Description=====
-Here we provide the characterization of this promoter in E. coli TOP10, which has a lacI genomic copy, constitutively expressed in a weak manner.
-The data below are referred to <partinfo>BBa_K173025</partinfo>, which is the measurement system of <partinfo>BBa_R0011</partinfo>.
-=====Characterization=====
-Compatibility: E. coli TOP10 in pSB1A2.
-<html>
-<table border=1 align="center" cellpadding=5px>
-<tr>
-<td rowspan=2 valign="bottom" align="center"><b>IPTG concentration<br></b></td>
-<td colspan=2 align="center"><b>LB</b></td>
-</tr>
-<tr>
-<td><b>Doubling time [minutes]</b></td>
-<td><b>RPU</b></td>
-</tr>
-<tr align="center">
-<td>0uM</td><td>     42 </td>
- <td>   1.52 [1.46; 1.59] </td>
-</tr><tr align="center">
-<td>10uM</td><td>     41 </td>
- <td>   1.83 [1.73; 1.90] </td>
-</tr><tr align="center">
-<td>50uM</td><td>     43 </td>
- <td>   1.78 [1.64 ; 1.92] </td>
-</tr><tr align="center">
-<td>100uM</td><td>     46 </td>
- <td>   1.73 [1.52; 1.95] </td>
-</tr><tr align="center">
-<td>500uM</td><td>     43 </td>
- <td>   1.61 [1.46 ; 1.81] </td>
-</tr><tr align="center">
-<td>1mM</td><td>     47 </td>
- <td>   1.78 [1.53; 2.01] </td>
-</tr><tr align="center">
-<td>2mM</td><td>     44 </td>
- <td>   1.47 [0.93; 2.05] </td>
-</tr>
-</table>
-</html>
-{|align="center"
-|[[Image:PV_A17_LB.png|700px|thumb|<partinfo>BBa_K173025</partinfo> Growth curves for BBa_K173025 in LB]]
-|-
-|[[Image:PV_A17_Scell_LB.png|700px|thumb|<partinfo>BBa_K173025</partinfo>(dGFP/dt)/O.D. in LB]]
-|-
-|[[Image:PV_A17_IndCurvLB.png|700px|thumb|<partinfo>BBa_K173025</partinfo> Induction curve of BBa_K173025 in LB]]
-|}
-=====Conclusions=====
-As TOP10 strains contains an expressed lacI in its genome, we wanted to know if it was enough to repress Plac activity in a high copy number plasmid in order to use it as a lactose/IPTG sensor. It is evident from induction curve that the behaviour of this part is almost independent from IPTG concentration, probably because lacI production by genomic DNA is too low. So our characterization has shown that <partinfo>BBa_R0011</partinfo> can be used as a strong constitutive promoter, with mean RPU higher than 1.5.
-Future work should be dedicated to the characterization of this BioBrick in low copy number plasmids and in strains containing lacIq mutation in order to buil-up new IPTG/lactose sensors.
-<div align="right">
-[[#top|Top]]
-</div>
-====='''Growth conditions'''=====
-======Microplate reader experiments======
-*8 ul of long term storage glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
-*The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
-*These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not comparable with the 1 cm pathlength cuvette) in a sufficient amount of medium to fill all the desired microplate wells.
-*These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (see [http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation Frame effect section] for details). All the wells were filled with a 200 ul volume.
-*If required, 2 ul of inducer were added to each single well.
-*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence (when required) and absorbance were measured with this automatic protocol:
-**37°C constant for all the experiment;
-**sampling time of 5 minutes;
-**fluorescence gain of 50;
-**O.D. filter was 600 nm;
-**GFP filters were 485nm (ex) / 540nm (em);
-**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting  before the measurements in order to make a homogeneous culture.
-**Variable experiment duration time (from 3 to 24 hours).
-====='''Data analysis''' =====
-======Growth curves======
-All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration).
-======Doubling time======
-The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line ''m''. Finally the doubling time was estimated as ''d''=ln(2)/''m'' [minutes].
-In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure.
-======Relative Promoter Units (RPUs)======
-The RPUs are standard units proposed by Kelly J. et al., 2008, in which the transcriptional strength of a promoter can be measured using a reference standard, just like the ground in electric circuits.
-RPUs have been computed as:
-[[Image:pv_rpu_formula.jpg|230px]]
-in which:
-*phi is the considered promoter and J23101 is the reference standard promoter (taken from Anderson Promoter Collection);
-*F is the blanked fluorescence of the culture, computed subtracting for each time sample fluorescence measure for negative control from that of culture, where the negative control is a non-fluorescent strain (in our experiment it is usually used TOP10 strain bearing <partinfo>BBa_B0032</partinfo> or <partinfo>BBa_B0033</partinfo>, which are symmply RBSs do not have expression systems for reporter genes);
-*ABS is the blanked absorbance (O.D.600) of the culture, computed as described in "Growth curves" section.
-RPU measurement has the following advantages (under suitable conditions)
-*it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
-*it uses a reference standard and so measurements can be compared between different laboratories.
-The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:
-*the reporter protein must have a half life higher than the experiment duration (we use GFPmut3, <partinfo>BBa_E0240</partinfo>, which has an estimated half life of at least 24 hours, and the experiments duration is always less than 7 hours);
-*strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
-*steady state must be valid, so (dF/dt)/ABS (proportional to the GFP synthesis rate per cell) must be constant.
-=====Inducible systems=====
-Every experiment is performed on the following cultures:
-*the culture of interest (system studied expressing GFP)
-*the benchmarck used to evaluate R.P.U. (<partinfo>BBa_K173001</partinfo> measurement part, that is <partinfo>BBa_J23101</partinfo> with <partinfo>BBa_E0240</partinfo> downstream)
-*a negative control (generally, <partinfo>BBa_B0033</partinfo> RBS)
-For inducible systems several plots are reported. The first plot is a panel containing 4 subplots, numerated this way:
-{|
-|(1)
-|(2)
-|-
-|(3)
-|}
-Plot (1) contains growth curves of the cultures, after blank value has been removed. Every curve is calculated averaging on three replicates of the same culture and subtracting the blank for each time sample. Blank is calculated averaging the replicates of blank wells.
-Plot (2) shows the logarithm of absorbance in exponential phase of bacterial growth, determined by a visual inspection of log-plots. These values are used to evaluate doubling time and R.P.U..
-Plot (3) contains (dGFP/dt)/O.D., the value named S_cell in Kelly J. et al., 2008 procedure for RPU evaluation.
-In these plots are reported black veritcal lines that define the range of values used to evaluate RPU. It is important to underline, as explained in next paragraph, that RPU are calculated on cultures at the same O.D. level, not at the same time.
-The second graphic shows S_cell VS O.D.. This plot allows the conparison of S_cell values between different cultures, that are supposed to reach the same level of growth not at the same time, but at the same O.D. value.
-The third graphic shows the induction curve. The RPU value is calculated on S_cell values corresponding to O.D. values in exponential phase (typically, from 0.05 to 0.16).
-The curve is obtained averaging in time S_cell values corresponding to exponential phase.
-Error bars rapresent the minimum and maximum value of R.P.U. belonging to the range of O.D. in exponential phase.
-====='''Materials'''=====
-*Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%
-*Antibiotics were: Ampicillin (Amp) 100 ug/ml stored at -20°C in 1000x stocks. Amp was dissolved in water.
-*LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.
-*M9 supplemented medium was prepared according to: [http://openwetware.org/wiki/Knight:M9_supplemented_media Openwetware protocol].
-*Ready made IPTG (Sigma) was stored at -20°C in a 200mM stock.
 ===User Reviews===
@@ Line 181: / Line 27: @@
 This review comes from the old result system and indicates that this part worked in some test.
 |}
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<I>UNIPV-Pavia iGEM 2009</I>
+|width='60%' valign='top'|
+[[Part:BBa R0011:Experience/iGEM09 UNIPV-Pavia|UNIPV-Pavia iGEM 2009's Experience]]: BBa_R0011 - Plac hybrid promoter
+|}
 {|width='80%' style='border:1px solid gray'
 |-
@@ Line 188: / Line 40: @@
 <I>UNIPV-Pavia iGEM 2010</I>
 |width='60%' valign='top'|
-__NOTOC__
+[[Part:BBa R0011:Experience/iGEM10 UNIPV-Pavia|UNIPV-Pavia iGEM 2010's Experience]]
-<partinfo>BBa_R0011</partinfo> hybrid lac promoter and the <partinfo>BBa_R0010</partinfo> wild type lac promoter were characterized at different copy number in TOP10 ''E. coli'' strain. This strain contains a lacI expression system in the genome.
+|}
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<I>Washington 2010</I>
+|width='60%' valign='top'|
+[[Part:BBa R0011:Experience/iGEM10 Washington|Washington 2010's Experience]]: R0011 in different plasmids
+|}
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_R0011 AddReview 4</partinfo>
+<I>iGEM Kyoto 2010</I>
+|width='60%' valign='top'|
+We measured RPUs of R0011 in low number copy plasmid with various concentrations of IPTG
-Induction static transfer function (computed in Relative Promoter Units), dynamics and metabolic burden were evaluated as a function of different IPTG concentrations in M9 supplemented with glycerol growth medium.
+[[Part:BBa_R0011:Experience/iGEM10_Kyoto|See details.]]
+|}
-A RFP generator (<partinfo>BBa_I13507</partinfo>) was used as a reporter gene. In particular, these measurement systems were used:
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_R0011 AddReview 4</partinfo>
+<I>iGEM11_Uppsala-Sweden</I>
+|width='60%' valign='top'|
+We measured RPUs of different promoters in low copy number plasmids.
-*<partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo>
+[[Part:BBa_R0011:Experience/iGEM11_Uppsala-Sweden|See details.]]
-*<partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>
+|};
-*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo>
-*<partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>
-At first, <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> inducibility was tested in a high copy number vector (<partinfo>pSB1A2</partinfo> or <partinfo>pSB1A3</partinfo>). The results are shown here as the relative RFP synthesis rate per cell.
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_I712019 AddReview 5</partinfo>
+<I>UT-Tokyo 2011</I>
+|width='60%' valign='top'|
+UT-Tokyo 2011 Team has characterized this part through the test of our [https://parts.igem.org/Part:BBa_K518002 Firefly-Renilla Dual Luciferase Assay Kit]. We succesfully evaluated the relative expression levels of [https://parts.igem.org/Part:BBa_R0011 BBa_R0011] with various IPTG concentration, compared to that of [https://parts.igem.org/Part:BBa_J23119 BBa_J23119] as a control. For experimental details, see [http://2011.igem.org/Team:UT-Tokyo our page].
+|};
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_K592008 AddReview 5</partinfo>
+<I>[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012]</I>
+|width='60%' valign='top'|
-{|align="center"
+'''iGEM Team Uppsala University 2012'''
-|[[Image:PV_hc_r001110LB.png|700px|thumb|Relative RFP synthesis rate per cell in <partinfo>pSB1A2</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB1A3</partinfo>-<partinfo>BBa_J04450</partinfo>. The error bars represent the standard errors of three independent measurements.]]
-|}
+'''Promoter strength'''
-Results show that in this condition <partinfo>BBa_R0010</partinfo> is about 2-fold stronger than <partinfo>BBa_R0011</partinfo>, but induced and uninduced cultures did not show differences in the RFP signal.
+[[Image:Promotertestuppsala2012.png|300px|thumb|Promoter strenghts in MG1566 and DH5alpha, relative to J23101.]]
-This result is expected because the vectors are propagated at about 200 copies per cell, while the lacI repressor is present at single copy in the genome and thus it is not able to repress the lac promoters in such high copy.
+A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (<partinfo>K864101</partinfo>) in the backbone <partinfo>K592200</partinfo> (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.
+Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.
-<partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> were then tested in the low copy (~5 copies per cell) vector <partinfo>pSB4C5</partinfo> in order to test their inducibility. The results are shown here as the RPU values at the steady state (constant RFP sysnthesis rate per cell) at different IPTG concentrations.
+{|
+|
+!MG1566
+!DH5alpha
+|-
+|J23101
+|1
+|1
+|-
+|J23106
+|0.19
+|0.37
+|-
+|J23110
+|0.27
+|0.50
+|-
+|J23116
+|N/A
+|0.11
+|-
+|Plac
+|0.34
+|0.67
+|-
+|PlacIq
+|0.03
+|0.05
+|-
+|T5lac
+|0.217
+|0.54
+|-
+|PLlacO
+|0.87
+|N/A
+|}
+The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.
+'''LacI repression'''
+The possibility of repression by lacI and induction by IPTG was evaluated in the pSB4C15Iq backbone. Cells grown with IPTG (0.5 mM) had a 100-fold increase of RFP expression, when compared to those grown without. Read about [https://parts.igem.org/Part:BBa_K864009:Experience pSB4C15Iq] for details.
-{|align="center"
-|[[Image:PV_rpu_r001110LB.png|700px|thumb|RPU of <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo> as a function of IPTG concentration. The error bars represent the standard errors of three independent measurements.]]
 |}
-Results show that in this condition both <partinfo>BBa_R0010</partinfo> and <partinfo>BBa_R0011</partinfo> produce different amounts of RFP as a function of the IPTG concentration. The amplitude of the two curves show that the promoters are very strong when induced with IPTG >= 10 uM. Although the experiments were carried out in the same conditions, the variability between experiments was high, especially for <partinfo>BBa_R0010</partinfo> (mean coefficient of variaton of about 37% for <partinfo>BBa_R0010</partinfo> and 15% for <partinfo>BBa_R0011</partinfo>), while the RPU variability between three wells in the same experiment is much lower (mean coefficient of variaton of bout 3.5% for both promoters).
-The above figure shows that <partinfo>BBa_R0011</partinfo> is stronger than the <partinfo>BBa_R0010</partinfo> wild type promoter in low copy plasmid. This result is unexpected because the same promoters in high copy vectors behaved differently (<partinfo>BBa_R0010</partinfo> was stronger than the <partinfo>BBa_R0011</partinfo>, see above).
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_K592008 AddReview 5</partinfo>
+<I>[http://2013.igem.org/Team:UCSF UCSF 2013]</I>
+|width='60%' valign='top'|
-In the uninduced state, <partinfo>BBa_R0011</partinfo> has about the same strength as the <partinfo>BBa_J23101</partinfo> reference standard promoter.
+<html>
-This static characteristic shows that the promoters are both leaky and a very low IPTG concentration (10 uM) is sufficient to trigger gene expression at *very* high levels.
+<regulartext1> The UCSF iGEM team tested the pLAC promoter (part BBa_R0011) by placing it in front of GFP and inducing with various concentrations of IPTG. We used this part in the design of our CRISPRi circuit and improved upon it through the following experiments:   <br>
-These results demonstrate that the genomic lacI is partially able to repress the two promoters, but very low IPTG concentrations are sufficient to bind the repressor and trigger the promoters transcription.
-'''Dynamic characterization in low copy vector:''' The figure below shows a typical relative RFP synthesis rate per cell time series for  <partinfo>BBa_J107010</partinfo> and <partinfo>BBa_J04450</partinfo> induced with 1 mM of IPTG and uninduced. These time series show that the full induction can be reached after about 50 min from the induction.
+<img align="center" style="margin-bottom:20px; width: 500px; margin-top:20px; padding:2; margin-left:55px;" src="https://static.igem.org/mediawiki/2013/8/85/PLAC_Time_Induction_Curve.png"> <br>
+Time curve for original pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader over time. GFP fluorescence were corrected for OD600 value. Error bars indicate standard deviation calculated on the basis of technical replicates.
-{|align="center"
-|[[Image:PV_dynamic_r001110LB.png|700px|thumb|Mean Scell signal as a function of time for <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J107010</partinfo> and <partinfo>pSB4C5</partinfo>-<partinfo>BBa_J04450</partinfo>. Induced (with 1 mM of IPTG) and uninduced cultures are shown. Induction occurs at t=0. The shown graph is relative to one of the three experiments performed in different days.]]
+<img align="center" style="margin-bottom:0px; width: 500px; margin-top:20px; padding:2; margin-left:55px;" src="https://static.igem.org/mediawiki/2013/9/9a/PLAC_dose_response.png"> <br>
+Dose-response curve for original pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.
+</html>
 |}
+{|width='80%' style='border:1px solid gray'
+|-
+|width='10%'|
+<partinfo>BBa_K2694000 AddReview 5</partinfo>
+<I>[http://2018.igem.org/Team:NDC-HighRiverAB NDC-HighRiverAB]</I>
+|width='60%' valign='top'|
+|
+<html>
+<b> QUALITATIVE DATA </b>
+<p>We first did a qualitative test. From the literature, we found that we could use 4-nitrophenyl palmitate and 4-nitrophenyl octanoate to test our esterase activity. Both substrates, when cleaved by an esterase, would go from clear to green, allowing us to monitor the cleavage activity visually. We set-up a time-course assay where we mixed 1mM of each nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer. We also tested DH5⍺ cells without any plasmid as well as the ester mixture with no cells as controls. <b>Figure 2</b> Shows the results from this experiment.</p>
+<p>From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.</p>
+<p> We did not add IPTG to these tests, so the green colour indicates that our promoter was "leaky", allowing continual expression of our EstA without the need of IPTG. </p>
+<img src="https://static.igem.org/mediawiki/2018/7/7e/T--NDC-HighRiverAB--4nitro.jpeg" alt="A comparison of 4-nitrophenyl palmitate and 4-nitrophenyl octanoate">
-'''Conclusion:''' the characterization of two IPTG-inducible promoters has been performed and the performance of these two promoters have been compared in terms of transcriptional strength. The reported results are easily sharable in different laboratories thanks to the used standard RPU approach.
+<div>
+  <p>Figure 2: The top row shows centrifuge tubes containing the 4-nitrophenol ester reaction mixture for the palmitate (a) and octanoate (b) esters with no cells added. The middle row shows the response for a negative control where DH5⍺ cells without any plasmid were added to reaction mixtures containing 4-nitrophenyl-palmitate (c) or 4-nitrophenyl-octanoate (d). The bottom row shows the response for the BBa_K2694000 circuit transformed into DH5⍺ cells when added to reaction mixtures containing 4-nitrophenyl-palmitate (e) or 4-nitrophenyl-octanoate (f). All reaction mixtures were 1mM of the nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer.</p>
+</div>
+<p>From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.</p>
-'''Methods:'''
+<b> QUANTITATIVE DATA </b>
-*A of long term storage glycerol stock was streaked on a LB plate with suitable antibiotic. Tha plate was incubated overnight at 37°C.
+<p>Once we had shown that our bacteria could cleave the substrates to some extent, we wanted to try to show this more quantitatively. In collaboration with the University of Calgary’s iGEM 2018 team, we designed an experiment to test our reaction in a time course, using their spectrophotometer, in order to more accurately determine the color change. </p>
-*A single colony was inoculated in 1 ml of M9 + suitable antibiotic in a 15 ml tube and incubated at 37°C, 220 rpm for about 16 hours.
+<p> <b>Graph 1</b> shows the averages of the results from all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl octanoate with and without IPTG. Our control started at 0 and ended at 0.002. All other samples increased for about 660 seconds and then started to level out. This graph shows that our part, BBa_k2694000 in <i> E.coli </i> was producing the EstA protein because the solution got progressively more green with the introduction of our bacteria, but without the bacteria the control did not change colour. Based on these averages, the IPTG did not appear to have an effect on our reaction under these conditions. Our bacteria passively produces the repressor for pLac, but since we added more pLac to the system, there may not have been enough of the repressor to inhibit the production of our EstA, which may explain why IPTG did not have any effect on the reaction. </p>
-*The grown cultures were then diluted 1:100 in 2-5 ml of M9 supplemented medium and incubated in the same conditions as before for about 4-5 hours.
+<img src="https://static.igem.org/mediawiki/2018/d/df/T--NDC-HighRiverAB--Graph2.jpeg" style="width:711px;height:400px;">
-*For each desired IPTG concentration to be tested, three 200 ul aliquots of the cultures were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame).
+<p class="center"> Graph 1: Average Absorbance Rates for the Breakdown of 4-Nitrophenyl Octanoate using BBa_K2694000 With and Without IPTG. </p>
-*2 ul of properly diluted IPTG (Sigma Aldrich) were added to the three wells for each desired concentration.
-*The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:
-**37°C constant for all the experiment;
-**sampling time of 5 minutes;
-**fluorescence gain of 50 or 80;
-**O.D. filter at 600 nm;
-**RFP filters at 535nm (ex) / 620nm (em);
-**15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
-**Experiment duration time: about 6 hours.
-*This experiment was performed three times in different days.
+<p> <b>Graph 2</b> shows the averages of the results from all our data for the absorbance values (measured at 400nm) for BBa_K2694000 breaking down 4-nitrophenyl palmitate with and without IPTG. Our control started at 0 and ended at 0.002. All other samples did not increase significantly. This shows that BBa_K2694000 is not good at breaking down the 4-nitrophenyl palmitate under these conditions. This further shows that IPTG did not have a noticeable effect on the expression of our gene. </p>
+<img src="https://static.igem.org/mediawiki/2018/c/c2/T--NDC-HighRiverAB--Graph4.jpeg" style="width:711px;height:400px;">
+<p class="center"> Graph 2: Average Absorbance Values for the Breakdown of 4-Nitrophenyl Palmitate by BBa_K2694000 With and Without IPTG. </p>
-'''Data analysis:''' Relative Promoter Units (RPUs) were estimated as described by [Kelly JR et al. (2009), J Biol Eng 3:4].
-Briefly:
-*Absorbance and fluorescence time series were normalized by subtracting the absorbance of the media and the fluorescence of a negative control (a non fluorescent TOP10 culture) respectively, thus yielding O.D.600 and RFP time series.
-*RFP synthesis rate per cell (called ''Scell'') was computed as (1/O.D.600)*dGFP/dt. (this signal is not actually the RFP synthesis rate, but is proportional to it).
-*The RFP synthesis rate per cell  was averaged at the steady state during the exponential growth phase (validated by identifying the linear region of the ln(O.D.600)).
+</html>
-*The RPU of the promoter of interest in a specific condition was computed as ''mean_Scell,phi/mean_Scell,J23101'' where phi is the promoter of interest, J23101 is the reference standard and ''mean_Scell'' is the mean Scell signal value, computed as explained above.
-|}
 <!-- DON'T DELETE --><partinfo>BBa_R0011 EndReviews</partinfo>