Difference between revisions of "Part:BBa K4030008"

Revision as of 02:20, 20 October 2021

Myc-HisA-OmpA-amilGFP-TT�

Profile

Name: Myc-HisA-OmpA-amilGFP-TT 

Base Pairs:4762 bp

Origin: E. coli , Acropora millepora , synthetic

Properties: Inducible expression amiGFP system

Usage and Biology

This chromoprotein from the coral Acropora millepora, amilGFP, naturally exhibits strong yellow color when expressed. The color is readily visible to naked eye both in LB-culture and on agar plates. Color development can be seen in less than 24 hours of incubation. We have successfully constructed an Arab-induced GFP expression system, which can control the intensity of GFP gene expression by changing the concentration of arabinose. The gene expression intensity of GFP can be regulated according to the intensity of the fluorescent signal. Using this expression system, we can achieve the regulated expression of various target genes in the future.

Figure 1. Schematic diagram of amiGFP inducible expression system..

Construct design

The amiGFP is under araBAD promoter, which is induced by arabinose. And amilGFP is linked with ompA and this sequence is inserted into plasmid (Figure 2 and 3).

Figure 2. Myc-HisA-OmpA-amilGFP-TT box..

Figure 3. Schematic map of amiGFP plasmid..

The profiles of every basic part are as follows:

BBa_K592010

Name: amilGFP

Base Pairs: 699bp

Origin: Acropora millepora, genome

Properties: This chromoprotein, naturally exhibits strong yellow color when expressed.

Usage and Biology

BBa_K592010 is a coding sequence of from Acropora millepora. It naturally exhibits strong yellow color when expressed and can be reported gene. The color is readily visible to naked eye both in LB-culture and on agar plates.

BBa_K4030010

Name: araBAD promoter

Base Pairs: 166bp

Origin: Escherichia coli

Properties: Inducible promoter used for protein expression

Usage and Biology

The araBAD promoter of the L-Arabinose operon of Escherichia coli allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli.

BBa_K4030000

Name: OmpA

Base Pairs: 63bp

Origin: Escherichia coli

Properties: Outer membrane protein A

Usage and Biology

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane.

BBa_K4030003

Name: Myc

Base Pairs: 30bp

Origin: Human

Properties: epitope tag

Usage and Biology

It is an epitope tag derived from c-myc gene

BBa_K4030006

Name: 6His

Base Pairs: 18bp

Origin: synthetic

Properties: Polyhistidine tag

Usage and Biology

It is a polyhistidine tag, which is used in the purification of recombinant proteins

Experimental approach

AraB expression plasmid and AmilGFP expression plasmid were transformed to E. coli Nissle 1917 by electroporation.

In the engineering cells simultaneously containing AraB expression plasmid and AmilGFP expression plasmid. The expression of in AmilGFP plasmid is to be induced by arabinose, which can be supplied by AraB encoded via the hydrolysis of araboxylan. So, for the sake of AmilGFP expression, araB expression plasmid must express active AraB. We tried to monitor the concentration of the reducing sugar arabinose and thus access the activity of AraB. The concentration of reducing sugar was determined by the DNS kit and the A540 were recorded accordingly.

Proof of function

(1) AmilGFP expression in cells transformed with plasmid C (pBAD-Myc-HisA-OmpA-amilGFP) In the cells transformed with plasmid puc57-kan-mini-J23101-OmpA-araB-TT (Plasmid A) the fluoroscopic data of cells increased with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with the prolongation of incubation time.

Figure 4. The fluoroscopic data of cells transformed with plasmid C with the addition of arabinose..

(2) AmilGFP expression in cells transformed with plasmids A and C In the cells transformed with plasmid A and C, the fluoroscopic data of cells increased with the addition of araboxylan. Meanwhile, the fluoroscopic data become larger with the prolongation of incubation time (Figure 5).

Figure 5. The fluoroscopic data of cells transformed with plasmids A and C..

Improvement of an existing part

Compared to the old part BBa_K592010, a medium-strength Plac promoter, we design a new part BBa_K4030008 which contains the araBAD promoter of the L-Arabinose operon of Escherichia coli allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli.

Peking iGEM 2016 has fused this part with triple spytag. The fused protein is participate in Peking’s polymer network. By adding this protein, the whole polymer network become visible in most conditions.

Based on the groups’ contribution, our team design the new composite part BBa_K4030008 to express AmilGFP regulated by pBAD promoter. We have successfully constructed an Arab-induced GFP expression system, which can control the intensity of GFP gene expression by changing the concentration of arabinose. The gene expression intensity of GFP can be regulated according to the intensity of the fluorescent signal. Using this expression system, we can achieve the regulated expression of various target genes in the future.

Figure 6 The blast results about the DNA sequence of our new part BBa_K4030008 and the old parts BBa_K592010..

First of all, we constructed a composite part Myc-HisA-OmpA-amilGFP-TT and transformed it into E.coli . We successfully expressed amilGFP protein induced by arabinose.

Furthermore, in order to test concentration of araboxylan, we set up different concentration of arabinose to test the fluorescence intensity. The experimental data shows that the optimal concentration of araboxylan is 3%.

In addition, our project can provide a system to express all kinds of useful genes. AmilGFP was successfully expressed, suggesting the feasibility and possibility of the inducible secretory expression of ClyR. Phage lyase ClyR has a broad bactericidal spectrum, especially the only one reported to be extremely strong against Streptococcus mutans and Streptococcus mulberry. The enzyme is promising to kill these two kinds of streptococci are the main cause of dental caries. Therefore, our engineering strain has a huge potential commercial market.

References

1,Yang, H., Linden, S. B., Wang, J., Yu, J., Nelson, D. C., & Wei, H. (2015). A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method. Scientific Reports, 5(1). https://doi.org/10.1038/srep17257

2,Xu, J., Yang, H., Bi, Y., Li, W., Wei, H., & Li, Y. (2018). Activity of the Chimeric Lysin ClyR against Common Gram-Positive Oral Microbes and Its Anticaries Efficacy in Rat Models. Viruses, 10(7), 380. https://doi.org/10.3390/v10070380\

3,Selwitz, R. H., Ismail, A. I., & Pitts, N. B. (2007). Dental caries. The Lancet, 369(9555), 51–59. https://doi.org/10.1016/s0140-6736(07)60031-2

4,Seo, E., Weibel, S., Wehkamp, J., & Oelschlaeger, T. A. (2012). Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins. International Journal of Medical Microbiology, 302(6), 276–287. https://doi.org/10.1016/j.ijmm.2012.05.002

5,Pitts, N. B., Zero, D. T., Marsh, P. D., Ekstrand, K., Weintraub, J. A., Ramos-Gomez, F., Tagami, J., Twetman, S., Tsakos, G., & Ismail, A. (2017). Dental caries. Nature Reviews Disease Primers, 3(1). https://doi.org/10.1038/nrdp.2017.30