Difference between revisions of "Part:BBa K1555000"

Latest revision as of 15:07, 4 October 2021

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

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 to construct BBa_K2555000. The construct include the promoter of cop A, ribosome binding site, self-cleaving RNA ribozyme RiboJ, and protein coding region-sfGFP. In E.coli. As a ATPase, cop A is regulated by a copper-responsive protein CueR(1). The promoter of cop A is more sensitive to copper than the other two copper-responsive promoter. RiboJ is an insulator commonly used in genetic circuits to prevent unexpected interactions between neighboring parts. Insulation with RiboJ may increase protein abundance. sfGFP increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during refolding(2). sfGFP has been proven to be very useful for improved protein detection(3).

Method

In order to show that our synthetic bacteria have a fully functional part that works, expression measurements were made. The construct was linked to sf GFP. For the parts plate reader was done. A plate reader is able to take optical density (OD600nm) and fluorescent measurements over time. OD is a measure of bacterial growth over time and fluorescence is a measure of protein expression over time.

Results

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The results of a plate reader experiment with different copper concentrations after five hours. Error bars show standard deviation of three repeats

We optimized BBa_k1555000 to be BBa_K2555000. The relative fluorescence intensity of BBa_K2555000 under different copper ion concentrations was higher than that of BBa_k1555000, and the increasing rate of relative fluorescence intensity was higher than that of BBa_k1555000.

References:

(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.

(2)Andrews BT, Schoenfish AR, Roy M, Waldo G and Jennings PA. The rough energy landscape of superfolder GFP is linked to the chromophore. J Mol Biol 2007;373: 476-490.

(3) Cabantous S and Waldo G. In vivo and in vitro protein solubility assays using split GFP. Nat Methods 2006; 3: 845-854.

Improvement of UI-Indonesia-2021 iGEM team

In the absence of a lab, Universitas Indonesia 2021 iGEM team offers literature that gives additional information about CopA promotor.

The function of CopA protein act as copper transporter and resistance. CopA transcription is regulated by CueR, a copper and silver responsive member of the MerR transcriptional activator family [1]. Escherichia coli Nissle 1917, which we used in our project, also has CueR in their genes.

CopA promotor also can sense gold ion concentration. The physical similarity between gold and copper affects metal binding site and activates pCopA. Cysteine Residues 112 and 120 are the main key of PcopA activation by copper or gold [1] The optimum response achieved with addition of 25 μm AuCl3 for mid-exponential phase cells. Higher concentration of gold causes toxicity for the cells. Half-maximal induction for CopA promotor at concentration ∼15.2 μm AuCl2 [1] 100 μm of bathocuproine disulfonate, cuprizone, and neocuproine (chelator for copper) does not affect gold activation for pCopA. Bathocuproine increases 9–34% of gold activation and cuprizone increases 42–45% of gold activation for pCopA; 200 μm EDTA (general chelator) can decrease the ability of gold activation for pCopA [1]

Previous study has reported that CopA promotor can sense ammonia in Bacillus licheniformis when CopA promotor sequence is inserted. A 0.0125% ammonium hydroxide was given to B. licheniformis whose original promotor was replaced by pCopA to express a-amylase. Result suggests that pCopA can mediate gene expression. Incubation after 96 h gives an optimal result (almost 5 fold of relative activity) for the a-amylase activity that we use and it remains stable until 120 h. Therefore, CopA promotor is also ammonia-inducible promoters [2]..

Reference:

(1) Stoyanov J, Brown N. The Escherichia coli Copper-responsivecopA Promoter Is Activated by Gold. Journal of Biological Chemistry. 2003;278(3):1407-1410.

(2) Shen P, Niu D, Permaul K, Tian K, Singh S, Wang Z. Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis. Journal of Industrial Microbiology and Biotechnology. 2021;48(5-6):1-8.