Team:HZAU-China/Help

Interaction work:

pCusC, PpcoA sequencing and promoter test

Author：Tian Liu

1. Experimental Background：

iGEM encourages the communication between teams and help others. So, when we heard HUST-China has something wrong about their promoter, we decided to give a hand. The one problem they had is they tried many times but failed to sequence those promoters in pET28a, and the other is they put mRFP under the promoter to do a promoter test but didn’t get result. So we got pCusC & PpcoA promoters with mRFP from them.

2. Proposes:

2.1 pCusC & PpcoA promoters sequencing

2.2 pCusC & PpcoA promoters test

3. Materials and equipment:

pCusC & PpcoA promoters with mRFP in pET28a plasmid
LB medium
Kanamycin
Multifunctional microplate reader
Shaking incubator

4. Method:

4.1 Sequencing

We design the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequencing the promoters. And Tsing Ke Biology Technology Company helped us synthetize the primer and sequencing those plasmids.

Analyzed the sample pCusC’s sequencing result, we find that the T7 promoter has not be digested , there is no pCusC in the plasmid, but mRFP has successful ligated. So we can observe the red is the red fluorescent protein expression by the T7 promoter. Therefore, in the subsequent promoter test, we abandoned the pCusC samples.

In the PpcoA samples we successfully found PpcoA and mRFP. However, at the end of PpcoA's sequencing result, there is more 6bp than HUST’s sequence. We take PpcoA promoter to detection.

4.2 Promoter test

a. Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Shaken overnight in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.

b. Add CuSO4 solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three copies for each concentration as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.

c. Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub as repetitions. Meanwhile, add LB culture medium containing and not containing 1.0mM CuSO4 as blank controls.

d. Read the OD₆₀₀ and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.

e. Divided the fluorescent intensity result using the value of OD₆₀₀. Record the data for compare and analysis.

5. Result and discussion

When we add the 1.0mM Cu to bacterium solution，we observed the color change of the visible to the naked eye. So we try to test the 1.0mM Cu in LB medium. Then we find its fluorescence value and the value of OD₆₀₀ with only LB samples have a large difference. So we decided to use the 1.0mM Cu in LB medium as the blank samples of the 1.0mM control.

We can observe from the figure, when the copper ion concentration is range of 0-0.1mM, with the increase of concentration of copper ion, promoter strength increased. In the copper ion concentration of 1mM, the promoter strength decreased.