Part:BBa_K2308002
sYFP2 knock In for fusion expression
This part is used to knock in the gene sYFP2 in Rhodobacter sphaeroides 2.4.1, replacing the end coden of gene puhA, and sYFP2 will be expressed with H subunit of light reaction center as fusion protein. The part is composed of 404bp of upstream DNA sequence(the end coden of puhA is not included) and 720bp coding sequence of sYFP2 (Part:BBa_K2308003) and 562bp of downstream DNA sequence. After homologous recombination, sYFP2 will be expressed with the H subunit through fusion expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 319
Illegal BamHI site found at 1217
Illegal XhoI site found at 96
Illegal XhoI site found at 159 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 36
Illegal NgoMIV site found at 54
Illegal NgoMIV site found at 336 - 1000COMPATIBLE WITH RFC[1000]
BBa_K2308002 is an important part in our experiment. It is composed of BBa_K2308006，BBa_K2308003 and BBa_K2308007. The function of this part is to carry out the homologous recombination on the genome of Rhodobacter sphaeroides 2.4.1.
In fusion experiment, sYFP2 was designed to be fused to the C-terminal of H subunit in the reaction center(RC) of Rhodobacter sphaeroides 2.4.1.
Figure 1：Modelling of RC-sYFP2 (GIF)
We constructed the plasmid using part BBa_K2308002, and after transformation and gene recombination, single clones are selected by antibiotics and PCR tests. In order to achieve gene knock out / knock in, we used a special plasmid called pDM4. The plasmid pDM4 contains gene Mob replicator oriV and gene SacB .Gene mob can be used to facilitate the use of bonding method of transformation. Replicator oriV couldn't replicate in the globular bacteria (Erythrocytes abscess λπ factor ) , thus single clones could be selected by chl resistance of the plasmid . Finally , through the gene SacB on pDM4 , sucrose was screened for secondary recombination .
Figure 2：Confirmed map of part BBa_K2308002
Figure 3：PCR test of BBa_K2308002
Fluorescence image shows sYFP2 which is fused with H submit of Rhodobacter sphaeroides 2.4.1 ’s reaction center folded correctly and was able to emit fluorescence.
Figure 4：Fluorescence image of whole cells of RC-SYFP2 cells when excited at 495nm
Figure 5：Absorption spectra of RC-sYFP2 strains compared with wild type.
The RC-sYFP2 fusion strain was created in a wild type background. Absorption spectrum of membranes from this strain recorded at room temperature show no isolated sYFP2 peak due to overlap with absorption of carotenoid B at 514 nm.
Figure 5：Growth rate of WT and WT RC-sYFP2.
The biomass of Rhodobacter sphaeroides 2.4.1 didn’t arise very much since the energy from sYFP2 is too little.
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