Part:BBa_K1555000

The copa promoter wth a gfp reporter

This part is our testing part.The copa is one of the candidates of our copper sensor.For comparing with other two sequences,marO（BBa_K1555001） and cueo（BBa_K1555002）,we linked it with a reporter(BBa_E0840).With the concentration of copper rises, the expression of downstream gfp coding gene increases.We designed two experiments to test those candidates' sensitivity and specificity individually by measuring fluorescence intensity.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 914
  

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

Experimental Design

Overview

CopA, the principal copper effluxATPase in Escherichia coli, is induced by elevated copper in the medium.[1] CopA promoter is active in the presence of copper ion.We intended to character copA promoter independently. Therefore , we utilized RiboJ which was placed between promoter and protein coding sequence to eliminate the interference of two different parts. Output ( fluorescence) depended only on the activity of copA promoter when be induced, and not the sequence at the part junction. RiboJ can reliably maintain relative promoter strengths.

First experiment:

We test copA promoter in BL21（DE3）,DH5α. By measuring fluorescence intensity in cells by flow cytometer,we got data to analyze sensitivity and specificity of copA promoter.

Results:

In our experiment, copA promoter was induced by different concentration of copper ion (37.5umol/L、50umol/L、62.5umol/L、75umol/L) . That fluorescence intensity in cell increase firstly and decreasewith small oscillations.（Fig.3A,B） At 4-5th hour fluorescence intensity in cell increases dramatically. Dose response curves was fitted to twice induction within 9 hours. CopA promoter has relative leaky basal expression by comparing the negative control’s output and basal leakage of copA promoter in E. coli expression systems(Fig.4). In comparison of two graphs A、B, we can obviously find that the degradation of protein is much faster in DH5α than that in BL21（DE3），because BL21（DE3 ） has a deficiency of protease. In the group of 0μmol/L Cu2+, the fluorescence shows a trend of falling firstly then rising（Fig.3C）. Actually, the fluorescence which produced by the leakage of copA will not change. The change quantity comes from the different growth periods of the E.coli. We added 40ul bacterial fluid into new medium with inductionto start measuring. So bacteria will go through a period of growing from growth period to maturation period, so as to the change of the fluorescence.Maturation period is great period for the expression of protein.

Fig.3.A.Changes in fluorescenceintensity induced by different concentration of copper ion in E.coli BL21 (DE3). B.Changes in fluorescence intensityinduced by differentconcentration of copper ion in E.coli DH5α. C .Changes in fluorescenceintensity in E.coli without induction comparing with two strains. D .Changes in fluorescenceintensity in E.coli which do not contain copA promoter comparing with two strains.

Fig.4.CopA promoter has leaky basal expression

Second experiment:

We placed insulator RiboJ between copA promoter and RBS.(Fig.5)

Fig.5.Two device used to detect copper in solution, the upper device has riboJ and the under one has no riboJ.

Result:

We use 50μmol/L copper ion to induce copA promoter.A device without RiboJ has an unstable Fluorescent quantity. At fourth hour, the fluorescence intensity in cells rose sharply. By contrast,a device with RiboJ response to copper ion and express GFP gradually. (Fig 6A) In addition,a device without RiboJ has high leakage with fluctuation. However, a device with RiboJ has low and stable leakage. (Fig 6B)

Fig .6 A.Changes in fluorescenceintensity induced by copper ion in E.coli over time B.leakage of copA promoter over time

Improvement: We concluded that RiboJ helps reduce the leakage of copA promoter greatly. After adding copper ions, the expression of green-fluorescent protein increased steadily. So, copA promoter with RiboJ can balance the expression of target protein in Escherichia coli.

Reference: [1] Outten FW, Outten CE, Hale J, O'Halloran TV. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. Journal of Biological Chemistry 2000;275:31024-9. ==Improvement of BFSU-China-2018 iGEM team

Experimental Design

We improve this by place Riboj between promoter and RBS, and replace sf GFP with GFP