Part:BBa_K4030008

Myc-HisA-OmpA-amilGFP-TT�

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 120
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

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.

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.

Usage and Biology

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

Usage and Biology

It is an epitope tag derived from c-myc gene

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.