Team:TJU China/Improve

Improve

Part:BBa_I15008 - heme oxygenase (ho1) from Synechocystis

Heme Oxygenase 1 (HO-1) is the one of two requisite genes required for the biosynthesis of phycocyanobilin from heme. For the past, this gene is almost used for generating biliverdin IXalpha, but BV produced is for phycocyanobilin (PCB) biosynthesis, which is the chromophore of some kinds of fluorescent proteins. This year our project is about smURFP, This novel far-red fluorescent protein evolved from APCα from Trichodesmium erythraeum can covalently attaches a biliverdin (BV) chromophore without a lyase, unlike its precursor APC which should use an auxiliary protein known as a lyase to incorporate phycocyanobilin. In addition, phycocyanobilin (PCB) is synthesized from BV, and PCB do not exists in mammals but BV does. So BV is better as a chromophore in some way, along with smURFP.

In addition, in order to make this part express better in hosts of our project (several intestinal bacteria), we optimize the codons of the part and submit another two new parts: Part:BBa_K2328003 for E.coli, EHEC, Citrobacter rodentium, Lactococcus Iactis, Bacaeroides fragilis, Enterococcus faecalis and Clostridium difficile and Part:BBa_K2328004 for Bifidobacterium longum. We do codon optimization for two obligate anaerobes just want to comfirm that this gene cannot work at all in obligate anarobes, which means HO-1 gene doesn't make any sense in these two bacteria, at least for now.

In our projext, we use HO-1 gene for two task: one is to produce BV in E.coli BL21, the other is to be a element in co-expression system (with fluorescent protein smURFP).

Results

I. BV production

We use this part to prduce Biliverdin (BV) in E.coli BL-21. To produce biliverdin，the gene of the HO-1 was connected to the expression vector---pET28bs and then transferred to target bacteria E.coli BL-21.The precursor of biliverdin-----HEME will be transferred to biliverdin with the help of HO-1.

To construct the co-expression system, the gene of fluorescent protein---smURFP and the gene of the HO-1 was connected to the same expression vector and then transferred to our target bacteria. The fluorescent protein will combine with biliverdin directly in our target bacteria and glow in the bacteria.

As shown in the picture, we can see the obvious change of medium in color.

Figure 1. The result after induction, the upper one is the control group, and the inferior one is the experimental group.

II. Co-expression with smURFP

a) Tests in vitro I

Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.

Table 1. Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.

b) Tests in vitro II

Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.

Figure 2. The result after induction, We can see the fluorescence emit from the whole body of the bacteria.

c) Tests in vivo

After tests in vitro, we used this engineered bacteria for experiments in vivo. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.

Figure 3. The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.