Difference between revisions of "Part:BBa K3182100"
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<partinfo>BBa_K3182100 short</partinfo>
 
<partinfo>BBa_K3182100 short</partinfo>
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This part is mainly about the pink-white screening method. For more information and characterization of the carbohydrate binding domain (CBDcipA) please see: <partinfo>BBa_K3182108/partinfo>.
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[[File:T--Linkoping_Sweden--fusionproteinillustration.jpg|420px|thumb|right|<b>Figure 1.</b> Mechanism of action for Novosite. The CBDcipA-fusion is attached to a polysaccaride material. By adding thrombin from any source the fusion protein will be cleaved and the C-terminal fusion protein will be released into the solution. By changing the fusion protein to an antimicrobial peptide/enzyme, and using the material as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.]]
 
[[File:T--Linkoping_Sweden--fusionproteinillustration.jpg|420px|thumb|right|<b>Figure 1.</b> Mechanism of action for Novosite. The CBDcipA-fusion is attached to a polysaccaride material. By adding thrombin from any source the fusion protein will be cleaved and the C-terminal fusion protein will be released into the solution. By changing the fusion protein to an antimicrobial peptide/enzyme, and using the material as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.]]
  
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<h2>Examples of biobricks assembled with this method</h2>
 
<h2>Examples of biobricks assembled with this method</h2>
 
<b>This method was used to assemble</b>: <partinfo>BBa_K3182103</partinfo>, <partinfo>BBa_K3182104</partinfo>, <partinfo>BBa_K3182105</partinfo>, <partinfo>BBa_K3182106</partinfo>, <partinfo>BBa_K3182107</partinfo>, <partinfo>BBa_K3182108</partinfo> into pSB1C3. However, as we quickly noticed, pSB1C3 was not able to replicate in <i>Vibrio natriegens</i> (Vmax). After countless failed transformation attempts, all the above parts were also assembled into a modified pUC19 vector using the pink-white screening method. The pUC19 variants were transformed without any problems into <i>V. natriegens</i> (Vmax). A total of 13 fusion proteins to the CBD was successfully ligated using this method.
 
<b>This method was used to assemble</b>: <partinfo>BBa_K3182103</partinfo>, <partinfo>BBa_K3182104</partinfo>, <partinfo>BBa_K3182105</partinfo>, <partinfo>BBa_K3182106</partinfo>, <partinfo>BBa_K3182107</partinfo>, <partinfo>BBa_K3182108</partinfo> into pSB1C3. However, as we quickly noticed, pSB1C3 was not able to replicate in <i>Vibrio natriegens</i> (Vmax). After countless failed transformation attempts, all the above parts were also assembled into a modified pUC19 vector using the pink-white screening method. The pUC19 variants were transformed without any problems into <i>V. natriegens</i> (Vmax). A total of 13 fusion proteins to the CBD was successfully ligated using this method.
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Revision as of 13:20, 17 October 2019

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 592
    Illegal NheI site found at 615
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

pT7-CBDcipA-pCons-AsPink This part is mainly about the pink-white screening method. For more information and characterization of the carbohydrate binding domain (CBDcipA) please see: No part name specified with partinfo tag.), seen in Figure 3, as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in Escherichia coli (E. coli) (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).

Figure 3. Benchling screenshot of the expression system. The T7-RNA-polymerase promotor is followed by a T7 g10 leader sequence which enhances the binding to the 16S ribosomal RNA. After the leader sequence a poly A spacer is found, which has been shown to increase translation in vitro. Before the start codon a strong RBS, g10-L, followed by an AT-rich spacer can be seen, which will slightly increase translation of the following gene.

Theoretical usage of this part

This part utilizes a pCons-AsPink dropout enabling colour-screening for positive colonies. Using BamHI and PstI or SpeI on both this part assembled in pSB1C3 (or vector of choice) and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.

Figure 4. The first plasmid (left) contains pCons-AsPink which results in pink colonies. When BamHI is used together with either SpeI or PstI pCons-AsPink is cut out (middle plasmid) and replaced with a compatible biobrick such as an antimicrobial agent (right plasmid). The colonies will then be white due to pCons-AsPink being cleaved from the plasmid. This results in a "pink-white screening".

Examples of biobricks assembled with this method

This method was used to assemble: BBa_K3182103, BBa_K3182104, BBa_K3182105, BBa_K3182106, BBa_K3182107, BBa_K3182108 into pSB1C3. However, as we quickly noticed, pSB1C3 was not able to replicate in Vibrio natriegens (Vmax). After countless failed transformation attempts, all the above parts were also assembled into a modified pUC19 vector using the pink-white screening method. The pUC19 variants were transformed without any problems into V. natriegens (Vmax). A total of 13 fusion proteins to the CBD was successfully ligated using this method.


Usage and Biology

Using Pink-White screening

Design any gene which you want to be fused to the CBDcipA with a BamHI recognition sequence in the 5'-end. The biobrick suffix can be used in the 3'-end. Cut the vector and insert with BamHI and PstI (SpeI also works), remove enzymes and mix, no need for gel purification. Using very high molar ratios might not yield any pink colonies at all, a molar ratio insert to vector of 7-20:1 will yield some pink colonies. Transform the host (BL21 (DE3) for quickest results) and incubate at 37 °C overnight, if the color is weak or can not be seen, incubate in 24-37 °C for an additional 16-24 hours.

Figure 5. (A,B) E. coli (BL21) cells used for pink-white screening after 16 hours in 37 °C. BBa_K3182100 was cut with BamHI and PstI to remove pCons-AsPink and BBa_K3182006 (magainin 2) or BBa_K3182104 (CHAP) was ligated into the plasmid. The white colonies indicate a successful ligation and the pink indicate unsuccessful ligation. (C) An agarose gel (1.3 %, TAE) with the biobrick BBa_K3182108 (CBD-sfGFP). Lane 1-3 contains three white colonies of CBD-sfGFP DNA amplified from pSB1C3 with VR and VF2. All fragment showed their expected size (1.2 kb). Lane 4 contains a 1 kb Plus DNA Ladder by New England Biolabs.

Results using Pink-white screening

Colonies from the pink-white screening were later sent for sequencing, where all white colonies came back as correctly ligated plasmids. The only colony-screened assembly attempt can be seen in Figure 5C, where all colonies screened showed the correct fragments, as well as all non-pink colonies showing green fluorescence (BBa_K3182108, sfGFP). After the results from Figure 5C had been sequenced, no more colony screenings had to be done. All white colonies sent to sequencing contained the correctly assembled fusion proteins.