Difference between revisions of "Part:BBa J23119:Experience"

No review score entered. Sriram Srikant

We used j23119 along with the RBS B0034 to provide a constitutive baseline expression during our characterization of p170 promoter cassette. To our suprise we found that under variable ph conditions the expression of the promoter varied, it showed extreme upregulation at low ph (~4.5).
For further details visit our wiki http://2010.igem.org/Team:IIT_Madras.

[http://2011.igem.org/Team:KULeuven K.U.Leuven iGEM 2011 Team]

Characterization by K.U.Leuven 2011 iGEM Team

We fused the constitutive promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose and IPTG. As such these results can serve as a control for the results obtained for the arabinose-inducible BBa_K584000 and IPTG-inducible BBa_K584002 bricks. We tested the activity in a TOP10F’ (Figure 1 & 3) as well as a MG1655 (Figure 2 & 4) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following website: [http://openwetware.org/wiki/E._coli_genotypes E.Coli_Genotypes].

Figure1

Figure2

Apart from a small growth defect and a somewhat lower fluorescence at the 8 hour time point after the addition of 2% arabinose, the activity of the constitutive promoter in TOP10F’ cells is unaffected by arabinose addition, as seen in Figure 1. For the MG1655 cells (Figure 2), however, we see clear effects of adding arabinose at concentrations of 0.2% or 2%, with promoter activities significantly lower than the control condition. This points to interference of high inducer levels with the activity of the promoter and the importance of metabolism of the inducer, as arabinose can be metabolized by MG1655 cells, but not by TOP10F’ cells.

Figure3

Figure3

For both TOP10F’ and MG1655 cells, the activity of the constitutive promoter is unaffected by the addition of IPTG (Figures 3 and 4).

Figure4

UNIQ697a85f8756cde80-partinfo-00000004-QINU

Experience of NAU-CHINA-2016 iGEM team--Kejian Shi

Experimental Design

To evaluate each site of the Anderson Promoter`s effect on the whole promoter, the NAU-CHINA Team used TASSEL5 to analyse the correlation degree between each mutation and the strength of the promoter.We combine the sequence and the data of strength , the P-Value is calculated using the GLM whose result is showed as the Manhadon. Our purpose is to add information which can help users to mutate the promoter to the suitable one for their teams.

Figure.1 P-Value by Sequence for Strength

Figure.1:It shows the correlation degree between the each position of the site of the promoter and the strength of the promoter using a series of Anderson promoters to analyse. The abscissa of the figure represents the position of the site of the sequence of the promoter. The position start at "0", so the last of the number of the site is "34". The ordinate of the figure represents the P-Value of the each site. The higher the P-Value is the higher effect of the site is,and the lower strength of the promoter is at the same time. For example ,we will find out that the 24th site reduce the strength of the promoter greatly from the figure.

Figure.2

Figure.2:The left list shows the position of each site of the promoter,the right list contains the P-Value of the each site. Some of the sites do not have the mutation so that TASSEL can not calculate their P-Value.

File:Strength data.xls File:Sequence data.xls

The two attachments is the sequence data and the strength data used in analysing with TASSEL.

Thessaly 2019 Characterization

Thessaly 2019 sought to characterize the coding sequence of TEM-optimized beta-lactamase (BBa_I757010) under the regulation of the constituve Anderson Family promoters BBa_J23100, BBa_J23105, BBa_J23106, BBa_J23119. Beta-lactamase is an enzyme that hydrolyses beta-lactams (e.g. ampicillin) and is naturally found in procaryotic cells. A colorimetric assay has been developed using nitrocefin as a substrate which after hydrolysis from beta-lactamase changes the reaction color, from yellow (380nm) to red (490nm).

To achieve that, the coding sequence was assembled with each promoter, a universal RBS (BBa_B0034) and a double terminator(BBa_B0015). The parts were cloned in pSB1C3 and pSB1K3 and transformed into E. coli DH5a competent cells. For protein expression, the plasmids were transformed into E. coli BL21 (DE3) competent cells.

For the beta-lactamase assay, we set up the following experimental design:

1. Grow BL21 (DE3) cells overnight in 5ml LB (~16h) at a shaken incubator, 37 degrees C / 210rpm

2. The following morning, measure the OD600 of overnight cultures

3. Dilute all cultures to OD600¬ = 0.05 in M9 minimal medium

4. Grow cells 37 degrees C /210 RPM until OD600=0.4-0.6 (~2h)

5. Dilute all cells to the same OD600 (e.g. 0.4)

6. Load 160 of culture in a 96-well plate (do triplicates). Add 40 ul 0.5 uM nitrocefin for a final concentration of 100nM

7. Measure the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) every 30 seconds for 25 minutes in a microplate reader. Shake between measurements.

To ensure that the absorbance shown corresponds only to enzymatic activity by beta-lactamase, we included 3 controls in the experiment. The first control has M9 medium only (no cells) and nitrocefin, the second has empty BL21 (DE3) cells (no plasmid) and nitrocefin, while the third has BL21 (DE3) cells containing the plasmid but not the part (empty plasmid). To obtain comparable results, we normalized all values by dividing OD490 by OD600.

The results are shown in the graph below