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

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.

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.

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.

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.

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