Difference between revisions of "Part:BBa K2328062"

Latest revision as of 09:42, 28 October 2017

HO-1 I (without a barcode)

The original part Part:BBa_I15008 has a barcode after coding sequence. So we submit a new part without the barcode for our composite parts. But we will upload all the data and infomation in both pages.

Sequence and Features

Usage

In order to fluoresce, smURFP must be combined with biliverdin (BV). HO-1 is the gene of the precursor of biliverdin. HO-1 can use the materials of the E.coil to produce biliverdin. So we want to construct a plasmid which can both express the smURFP gene and HO-1 gene. Through this construction, we can achieve the co-expression in the E.coil. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil to produce fluorescence under the wavelength of 642 nm without adding BV additionally. Besides, this is a codon-optimized version for higher HO-1 expression in Escherichia coli.

Biology

One of our methods is co-expression. Because the HO-1 needs to use oxygen to produce BV, it is adoptable in E.coil which is a kind of facultative anaerobic bacteria. And the HO-1 gene is from the Block Library. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil and we can achieve the co-expression in the E.coil.

Reference

[1] Dong Chen , Jason D Brown , Yukie Kawasaki , Jerry Bommer and Jon Y Takemoto . Scalable production of biliverdin IXα by Escherichia coli. [J].BMC Biotechnology, 2012.

[2] Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016 Sep;13(9):763-9.

[3] heme oxygenase (ho1) from Synechocystis Part:BBa_I15008

[4] HO-1 II codon-optimized for intestinal bacteria Part:BBa_K2328003

[5] HO-1 III codon-optimized for Bifidobacterium longum Part:BBa_K2328004

Results

I. BV production

We use this part to prduce Biliverdin (BV) in E.coli BL-21. As shown in the picture, we can see the obvious change if 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

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.

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, the upper one is the control group, and the inferior one is the experimental group.

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

For other experiments for some intestinal bacteria, we use our codon-optimized gene to express HO-1 in facultative anarobes.