Difference between revisions of "Part:BBa K3739042"
Line 3: Line 3:
 
<partinfo>BBa_K3739042 short</partinfo>
 
<partinfo>BBa_K3739042 short</partinfo>
  
−
To let the mussel stick to a solid surface.
+
It is a kind of mucin from mussels, including AKTA expression tags and 6xHis affinity tags which can let the mucin purified by tags.
 +
 
 +
===Biology===
 +
Mfp5 is a kind of mucin from mussel, which is one of the main proteins in adhesion interface. It’s side chain contains a lot of dopamine and is the main factor of adhesion.
 +
 
 +
===Usage===
 +
By codon optimization and adding a 6His-tag, the sequence suitable for expression in BL21(DE3) was constructed. The coding sequence of target gene was inserted into the expression vector with T7 promoter to obtain BBa_, and the constructed plasmid was transformed into BL21(DE3) to verify its expression.
 +
Figure 2 Gene circuit diagram of PPO
 +
===Characterization===
 +
====1. Identification====
 +
After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Figure 3.
 +
====2. Purification and Proof of the expression====
 +
T7 promoter was used in the composite part BBa_ to express AKTA-his-Mfp5 in BL21(DE3). Then we used GE-AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein. And then with tyrosinase modification, we get sticky proteins.  Then, our target bands are obserbed through SDS-PAGE and the result is shown in figure 4.
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->

Revision as of 17:03, 20 October 2021


J23100-B0030-AKTA-his-Mfp5-B0010

It is a kind of mucin from mussels, including AKTA expression tags and 6xHis affinity tags which can let the mucin purified by tags.

Biology

Mfp5 is a kind of mucin from mussel, which is one of the main proteins in adhesion interface. It’s side chain contains a lot of dopamine and is the main factor of adhesion.

Usage

By codon optimization and adding a 6His-tag, the sequence suitable for expression in BL21(DE3) was constructed. The coding sequence of target gene was inserted into the expression vector with T7 promoter to obtain BBa_, and the constructed plasmid was transformed into BL21(DE3) to verify its expression. Figure 2 Gene circuit diagram of PPO

Characterization

1. Identification

After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Figure 3.

2. Purification and Proof of the expression

T7 promoter was used in the composite part BBa_ to express AKTA-his-Mfp5 in BL21(DE3). Then we used GE-AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein. And then with tyrosinase modification, we get sticky proteins. Then, our target bands are obserbed through SDS-PAGE and the result is shown in figure 4.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]