Part:BBa_K4093006

pSIP403-PUS-xyn A

pSIP403-PUS-xyn A

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

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.

Figure 1. Schematic maps of pSIP403-PUS-xynA.

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

The profiles of every basic part are as follows:

BBa_K4093003

Name: pSIP403 Base Pairs: 7435bp Origin: Bacillus subtilis Properties: a plasmid vector for protein expression in L. reuteri

Usage and Biology

BBa_K4093003 is a plasmid vector for protein expression in L. reuteri.

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 3. electrophoregram of XynA.

Figure 3 is about the electrolysis of XynA. The result of electrolysis can clearly show whether the XynA is successfully amplified. XynA was amplified by PCR. The length of XynA is 650 bp. By compared the marker and the place of DNA, the PCR of XynA is successful.

Figure 4. electrophoregram of enzyme-digested products of pSIP403 and PUS Channel 1~2: pSIP403, 6kbp, correct Channel 3~4: PUS, 300bp, correct.

Figure 5. Colony PCR results of Lactobacillus reuteri/pSIP403-PUS-xynA and marker maps It can be clearly seen that our plasmid pSIP403-PUS-xynA has been successfully transformed into L. reuteri.

Proof of function

A. Secretory expression of pSIP403-PUS-xyn A 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 6.

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 7. Standard curve for reducing sugar.

Table 2. Calculation of average unit enzyme activity.

Figure 8.

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.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 7435
  Illegal PstI site found at 7668
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 7668
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 414
  Illegal BamHI site found at 4177
  Illegal XhoI site found at 2390
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 7435
  Illegal PstI site found at 7668
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 7435
  Illegal PstI site found at 7668
  Illegal AgeI site found at 420
  
• 1000
  COMPATIBLE WITH RFC[1000]