Difference between revisions of "Part:BBa K4093004"

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pMD19-T-xynA

pMD19-T-xynA

Profile

Name: pMD19-T-xynA

Base Pairs: 3346bp

Origin: E. coli , Bacillus subtilis ,synthetic

Properties: Produce recombinant xylanase in E.coli.

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

Plasmids pMD19-T-xynA is constructed to produce recombinant xylanase in E. coli (Figure 1).

Figure 1. Schematic map of pMD19-T-xynA.

Figure 2. The workflow of constructing pMD19-T-xynA.

The profiles of every basic part are as follows:

BBa_K4093002

Name: pMD19-T Base Pairs: 2704bp Origin: purchase from Takara Properties: a plasmid vector for protein expression in E.coli

Usage and Biology

BBa_K4093002 is a linearized vector with a single 3’-terminal thymidine at both ends. The T-overhang ends at the cloning site improve the efficiency of ligation of PCR products which contain A-overhangs at 3’-ends.

BBa_K4093000

Name: xynA Base Pairs: 642bp Origin: Bacillus subtilis, synthetic 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.

Experimental approach

Figure 3. electrophoregram 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. The electrophoresis results of pMD19-T-xynA enzyme digestion by NheI and HindIII. Channel 1: pMD19-T-xynA-LCX-ZZY, correct. Channel 2: pMD19-T-xynA-HHJ, correct.

The pMD19-T-backbone and xynA-fragment were connected by T4 ligase. Finally, we did double enzyme digestion by NheI and HindIII and sequenced for recombinant plasmid pMD19-T-xynA.

Figure 5. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of pMD19-T-xynA.

According to the sequencing results (Fig.3) from the sequencing company, the solid black line is the ideal state of PMD19-T-XynA and the DNA sequence of our plasmid matches the profile. We got correct plasmid.

Proof of function

Figure 6. SDS PAGE result of BL21(DE3)/pMD19-xynA, Coomassie blue staining.

After the sample has been centrifuged and sonicated. we put it to SDS-PAGE(SDS-polyacrylamide gel electrophoresis) which could determine the level of protein expression. As seen in the gel map (Maker, culture solution, intracellular supernatant, intracellular precipitation), the second red line of Marker represents 25KDa and the target protein should be 23.28kDa(Fig. 5). The blue line in the culture solution was near 23 KDa, it indicates that our protein could be sucessfully expressed in E. coli.

Figure 7. DNS test results of E. coli/pMD19-xynA.

The enzyme activity test results showed groups from the left to the right, respectively: blank, culture medium supernatant * 2, intracellular supernatant * 2, and intracellular precipitation * 2. It could be seen by naked eyes that in the seven bottles of solution, the culture medium solution showed red after DNS reaction, which indicated that we had xylanase with well enzyme activity to degrade xylan and this result is consistent with the SDS PAGE result.

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