Part:BBa_K4093005

PUS-xyn A

PUS-xyn A

Profile

Name: PUS-xynA Base Pairs: 936bp Origin: E. coli , Bacillus subtilis ,synthetic Properties: Produce recombinant xylanase with secreted protein PUS

Usage and Biology

Feed grains are whole grains such as corn, wheat and barley used in the feeding of livestock and poultry. At present, corn is the main fodder in the feed industry. Due to the increasing shortage of raw materials and the rising price, the development of the feed industry has been greatly limited. One of the important measures to alleviate the shortages of corn is to fully develop wheat, grain, and bran, which are abundant in China, to replace corn. However, the cell walls of cereals such as wheat, cereal, and bran contain anti-nutritional factors such as arabinoxylan and non-starch polysaccharide (NSP), which will affect the digestibility of nutrients in single-stomach animals and the absorption of nutrients in poultry. Arabinoxylan is a polysaccharide found in rice bran (hemicellulose B) edible fiber. Therefore, we decide to aim at adding xylanase to the feed to degrade arabinoxylan, so as to improve the feed absorption efficiency.

Construct design

Figure 1. Design diagram.

Our project is to design an edible "drink" for monogastric animals, mainly poultry through biosynthesis technology. The core product will be a probiotic that can produce xylanase. In this project, we designed to construct a plasmid expressing xylanase gene, then we transformed them into Escherichia coli and Lactobacillus reuteri for further performance analysis. Compared to traditional feed, our drink contains the probiotics that could help poultry digest xylan, thus increasing feed efficiency. In this way, not only can the time and economic cost of feeding be saved, but also the gastrointestinal tract of the animal can be protected and thus the disease rate can be reduced. The profiles of every basic part are as follows:

BBa_K4093000

Name: xynA Base Pairs: 642bp Origin: Bacillus subtilis Properties: endo-1,4-beta-xylanase

Usage and Biology

BBa_K4093000 is a coding sequence of from Bacillus subtilis. Xylanase is a kind of complex enzyme preparation specialized in degrading xylan in cereals.

BBa_K4093001

Name: PUS Base Pairs: 294bp Origin: lactic acid bacteria Properties: Secreted protein

Usage and Biology

BBa_K4093001 is a coding sequence of lactic acid bacteria, which can help protein to be secreted outside the cell.

Experimental approach

Figure 2. The workflow of constructing pSIP403-PUS-xynA.

Proof of function

A. Secretory expression of pSIP403-PUS-xynA in L. reuteri. At this time, start induction. The recombinant strain is added with 25 ng/mL SppIP to induce expression. After shaking at 37°C and 220r/ml for 3 hours, samples are collected at different time points to establish a function model of time and secreted expression; Take 20ml of the recombinant strain after induction culture, of which 1ml is used to detect the OD600 of the sample with an ultraviolet spectrophotometer.

Figure 3.

B．DNS enzyme activity detection Plasmid pSIP403-PUS-xynA transformed into L. reuteri to for secretory expression of xynA in L. reuteri. As shown in Table 1, significant xylanase activity was detected from broken supernatant by DNA method.

Table 1. Xylanase activity (OD540 nm) was detected from broken supernatant by DNA method.

A standard curve for reducing sugar was prepared using glucose (Figure 7).

Figure 4. Standard curve for reducing sugar.

Table 2. Calculation of average unit enzyme activity.

Figure 5.

The DNS color method was used to detect the unit enzyme activity of two parallel group samples at different induction time points, and the average unit enzyme activity was calculated. The experimental data showed that the enzyme activity was maximum when 25 ng/mL SppIP was induced for 8-12 hours. Is the best induction time.

Improvement of an existing part

According to the registry, the part BBa_K1175005 designed by group iGEM13_WLC-Milwaukee, described a  endo-1,4-beta-xylanase gene xynA. And there was xylanase enzyme activity data in support of its function. In 2014, iGEM team Heidelberg improved this part by removing the first 84 basepairs and adding a Ribosome Binding Site thus making the gene product (xylanase) retainable in the cytoplasm in E. coli. They characterize their part BBa_K1362020 in respect to its thermal stability by conducted a Heatshock Assay. Their results indicated a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C. In 2015, iGEM team TU_Darmstad further improved the initial part BBa_K1175005 by adding a linker-ligand sequence to fuse the protein to an in vitro scaffold for easy purification and increasing the degradation efficiency of xylan (BBa_K1602035). However, they just exemplarily the overexpression of degradation enzyme Acetyl Esterase fused to a linker binding the SH3 domain of the protein scaffold without enzymes activity. Compared to the above parts BBa_K1175005, BBa_K1362020 and BBa_K1602035, we improved the sequence of xynA and added a coding sequence of lactic acid bacteria, which can help xylanase to be secreted outside the cell.

Figure 6. The blast results about the DNA sequence of our new part BBa_K4093005 and the old parts BBa_K1175005, BBa_K1362020 and BBa_K1602035.

Besides, our results show that broken supernatant presents an obvious enzyme activity than that of blank control indicate recombinant protein secret outside cell successfully. In addition, we will establish a business development department aiming at an edible "drink" that can produce xylanase for monogastric animals. This feed additives or liquid beverage and can be sold to poultry breeders.

Future plan

We aim at developing a probiotic (Lactobacillus) containing xylanase to produce feed additives and poultry beverages, which are believed to help poultry digest xylan and enhance their health. Maybe it is possible for us to produce a novel “mixed feed” that combines other feed additives to enhance more poultry and ruminants’ digestion. After in-depth research, we obtained a feasible plan of “mixed feed” and the details of this plan can be seen at our wiki page partnership.

References

1.Beasley S S,Takala T M， Reunanen J， Apajalahti J， Saris P E.2004.Characterization and electrotransformation of Lactobacillus crispatus isolated from chicken crop and intestine. Poult Science，83(1)：45-48; 2.De Vos W M.1999.Gene expression systems for lactic acid bacteria. Current Opinion in Microbiology，2(3)：289-295; 3.Silversides F G， Scott T A， Korver D R，Afsharmanesh M，Hruby M.2006. A study on the interaction of xylanase and phytase enzymes in wheat-based diets fed to commercial white and brown egg laying hens.Poultry Science，85(2)：297-305; 4.崔罗生，祝茂生，徐顺清，朱辉，梁运祥，张忠明.2009.黑曲霉木聚糖酶基因(xyn A) 在大肠杆菌中的表达及酶学分析.华中农业大学学报，28(1)：48-53； 5.李慧.2010. 蛋白酶和木聚糖酶对肉鸡生长性能，消化机能及血液指标的影响.[研究生学位论文].杨凌：西北农林科技大学； 6.https://wenku.baidu.com/view/bb1a6d76590216fc700abb68a98271fe910eafb9.html

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]