Difference between revisions of "Part:BBa K4099001"

 
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<partinfo>BBa_K4099001 short</partinfo>
 
<partinfo>BBa_K4099001 short</partinfo>
  
−
pNCas9
+
=== Profile ===
 +
==== Name: pNCas9 ====
 +
==== Base Pairs: 4107bp ====
 +
==== Origin: Streptococcus pyogenes, Addgene ====
 +
==== Properties: A dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system ====
 +
 
 +
=== Usage and Biology ===
 +
BBa_K4099001 is a coding sequence of Cas9, an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence
 +
 
 +
[[File:T--Shanghai HS ID--BBa K4099000-Figure1.png|500px|thumb|center|Figure1. Principle diagram of CRISPR-Cas9..]]
 +
 
 +
=== Experimental approach ===
 +
We connected the optimized pNCas9 with pLCNICK vector. And then we use the homologous combination to combine pLCNICK with sgRNA and HA to get recombinant plasmidpNCas9-LSEI-2094.
 +
=== Verification result of colony PCR ===
 +
 
 +
[[File:T--Shanghai HS ID--BBa K4099001-Figure6.png|500px|thumb|center|Figure 2. PCR verification result..]]
 +
 
 +
As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are consistent with the DNA profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the plasmid in E. coli.
 +
 
 +
=== Proof of function ===
 +
After we electrotransformed the plasmid pIB165 as the foreign DNA to test the transformation efficiency of our modified L. casei, it took several days to culture and finally saw the comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation efficiency with remarkably more colonies than the wild (Wild).
 +
 
 +
1.Transformation test result
 +
[[File:T--Shanghai HS ID--BBa K4099000-Figure2.png|500px|thumb|center|Figure 3. Comparison between the wild L. casei (left) and modified L. casei (right, LSEI-2094 knocked out) in transformation..]]
 +
 
 +
Graph 1. Comparison between the wild L. casei and modified L. casei in transformation
 +
 
 +
[[File:T--Shanghai HS ID--BBa K4099000-Figure3.png|500px|thumb|center|Figure 4. Histogram comparison between wild strain and KO strain..]]
 +
The profiles of every basic part are as follows:
 +
In addition, we measured OD600 of these strain groups which were pre-spread plates with different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig. 8).
 +
In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas.
 +
 
 +
==== References ====
 +
==== 1.Roberts RJ (November 1976). "Restriction endonucleases". CRC Critical Reviews in Biochemistry. 4 (2): 123–64. ====
 +
==== 2.Kessler C, Manta V (August 1990). "Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)". Gene. 92 (1–2): 1–248. doi:10.1016/0378-1119(90)90486-B. ====
 +
==== 3.Pingoud A, Alves J, Geiger R (1993). "Chapter 8: Restriction Enzymes". In Burrell M (ed.). Enzymes of Molecular Biology. Methods of Molecular Biology. 16. Totowa, NJ: Humana Press. pp. 107–200. ====
 +
==== 4.Arber W, Linn S (1969). "DNA modification and restriction". Annual Review of Biochemistry. 38: 467–500. ====
 +
==== 5.Krüger DH, Bickle TA (September 1983). "Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts". Microbiological Reviews. 47 (3): 345–60. ====
 +
==== 6.Kobayashi I (September 2001). "Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution". Nucleic Acids Research. 29 (18): 3742–56. ====
 +
==== 7.汪川 & 张朝武.(2008).以益生菌为载体的基因工程疫苗研究进展. 卫生研究(01),118-122. doi:CNKI:SUN:WSYJ.0.2008-01-045. ====
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 06:35, 19 October 2021


pNCas9

Profile

Name: pNCas9

Base Pairs: 4107bp

Origin: Streptococcus pyogenes, Addgene

Properties: A dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system

Usage and Biology

BBa_K4099001 is a coding sequence of Cas9, an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence

Figure1. Principle diagram of CRISPR-Cas9..

Experimental approach

We connected the optimized pNCas9 with pLCNICK vector. And then we use the homologous combination to combine pLCNICK with sgRNA and HA to get recombinant plasmidpNCas9-LSEI-2094.

Verification result of colony PCR

Figure 2. PCR verification result..

As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are consistent with the DNA profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the plasmid in E. coli.

Proof of function

After we electrotransformed the plasmid pIB165 as the foreign DNA to test the transformation efficiency of our modified L. casei, it took several days to culture and finally saw the comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation efficiency with remarkably more colonies than the wild (Wild).

1.Transformation test result

Figure 3. Comparison between the wild L. casei (left) and modified L. casei (right, LSEI-2094 knocked out) in transformation..

Graph 1. Comparison between the wild L. casei and modified L. casei in transformation

Figure 4. Histogram comparison between wild strain and KO strain..

The profiles of every basic part are as follows: In addition, we measured OD600 of these strain groups which were pre-spread plates with different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig. 8). In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas.

References

1.Roberts RJ (November 1976). "Restriction endonucleases". CRC Critical Reviews in Biochemistry. 4 (2): 123–64.

2.Kessler C, Manta V (August 1990). "Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)". Gene. 92 (1–2): 1–248. doi:10.1016/0378-1119(90)90486-B.

3.Pingoud A, Alves J, Geiger R (1993). "Chapter 8: Restriction Enzymes". In Burrell M (ed.). Enzymes of Molecular Biology. Methods of Molecular Biology. 16. Totowa, NJ: Humana Press. pp. 107–200.

4.Arber W, Linn S (1969). "DNA modification and restriction". Annual Review of Biochemistry. 38: 467–500.

5.Krüger DH, Bickle TA (September 1983). "Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts". Microbiological Reviews. 47 (3): 345–60.

6.Kobayashi I (September 2001). "Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution". Nucleic Acids Research. 29 (18): 3742–56.

7.汪川 & 张朝武.(2008).以益生菌为载体的基因工程疫苗研究进展. 卫生研究(01),118-122. doi:CNKI:SUN:WSYJ.0.2008-01-045.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3511
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3304
  • 1000
    COMPATIBLE WITH RFC[1000]