Connectee:

Schematic introduction

As mentioned in the overview, in order for the protein to bind to the plasmid–the connector, we have designed two sorts of delicat fusion proteins–the connectee. The first kind of connectee is free while the second is anchored to the cell membrane. Both of them consist of various sections, which are shown in the schematic diagrams below.

1. TAL protein, the transactivator-like protein. According to its special structure, the TAL protein plays a great role in DNA binding. The 2012 Freiburg iGEM team has offered us a whole set of 96 TAL-protein direpeat bioparts, with which we are supposed to build functional TAL proteins. Since each TAL protein can identify a 14-nucleotide target sequence, the first and fourteenth nucleotide being Thymin, all the 96 parts can be used to identify more than 16 million different nucleotide sequences, which makes it very convenient for us to choose a sequence for the fusion protein to bind to.
2. The enzyme we want to bring together. Different proteins that bind to the same plasmid contain different enzymes from the same metabolic pathway.
   With these two parts combined by a flexible linker, we can obtain the first type of connectee, that is free from the cell membrane. But if we want to bind the fusion protein to it, at least two more sections are needed, which are listed below.
3. SsDsbA, a signal peptide at the N-terminal of the protein. It can direct the fusion protein to the preiplasm.
4. Lgt, a transmembrane protein whose function has been identified in previous iGEM projects. All these four sections, together with linkers, enable us to build the second type of fusion protein, which can be anchored to the cell membrane. Besides, FP, the fluorescent protein is also needed when we want to detect whether the fusion protein is expressed. During detection, FP is expressed between ssDsbA and Lgt while enzyme is not linked to the whole fusion protein in order to ensure its proper size. For different connectee, we can use different fluorescent protein to detect the expression successively, so that they can be distinguished easily.
   In conclusion, with the help of our artificial multi-enzyme complex systems, we are sure to improve the dynamic characteristics of metabolic reactions in many applicational fields.

As mentioned earlier, TAL effectors can bind DNA with target sequence. We devote to apply this binding character on the plasmid to achieve enzyme polymerization in vivo.

At first, test should be taken to check whether TAL can bind plasmid DNA in prokaryotic system, here we use E.coli. We designed two kinds of fusion protein called connectee.

Connectee 1:

The first kind of connectee is designed to be tested on the membrane, which is consist of three major domains, membrane anchor system(ssDsbA-Lgt), mRFP and TAL effectors.

The reason why we choose a membrane anchor is that:

1. TAL could bind nucleoid which may bring some effect on bacteria growth.
2. Membrane scaffold is a natural scaffold.
3. Exogenous proteins often form inactive inclusion body when expressed in the prokaryotic system

The membrane anchor system (ssDsbA-Lgt) comes from BBa_K771000 designed by iGEM12_SJTU-BioX-Shanghai(reference); mRFP comes from BBa_E1010 designed by Antiquity; TAL effectors comes from TAL-Protein DiRepeats (Bba_K747000 to Bba_K747095) designed by iGEM12_Freiburg. We use Helical Linker to connect mRFP and Lgt, in accord with iGEM12_SJTU-BioX-Shanghai. While in consideration of any possible stereospecific blockade when TAL binds plasmid DNA, we choose Flexible Linker to connect Lgt and TAL.

Connectee 2:

The second kind of connectee is designed to be tested in the cytoplasm, which only contain TAL effectors.

Connector:

As iGEM12_Freiburg designed, we can choose a 14-nucleotide-long Transactivator-like (TAL) protein TXXXXXXXXXXXXT to recognize the same sequence on the plasmid DNA.
The plasmid here we called connector.

The principle for choose a TAL recognize sequence:

1. Not exist in expression vector;
2. Not exist in the sequence of connectee;

Test method:

In consideration of our multiple-enzyme system may be applied in the following experiment, we choose pRSFDuet–1(NOVAGEN) as the expression vector.

pBluescript II KS(+) is chose as the connector for test for several reasons:

1. High copy number;
2. Medium length—2961bp
3. Easy to test whether binding a TAL may have effect on gene expression—lacI & blue-white spot screening

[图片四右]

Similar with the cross-linked ChIP, we use formaldehyde to cross-link connectee and connector

After that we do immunoprecipitation to get the protein-plasmid complex and digest protein

Finally, doing a PCR to check is there any plasmid DNA exist.

A convenient part:

In order to be used cooperatively with TAL-Protein DiRepeats (Bba_K747000 to Bba_K747095), we also design a part named ssDsbA-mRFP-Lgt-TAL adapter-His Tag (BBa_K1453000).

This part is consists of T1 sequence, T14 sequence and two sites for type II restriction enzyme BsmBI. When digested with BsmBI, this part can produce two sticky-ends. One can complement with the first TAL-Protein DiRepeat (Bba_K747000 to Bba_K747015) at 5’ of the sequence, while the other can complement with the sixth TAL-Protein DiRepeat (Bba_K747080 to Bba_K747095) at 3’.

With the rest of TAL-Protein DiRepeats (Bba_K747016 to Bba_K747079), users can synthesize a 14-nucleotide-long Transactivator-like (TAL) protein to recogize their own connector and design their own polymerization. (Golden Gate Cloning)

More details about membrane anchor, please view this page.

More details about TAL and Golden Gate Cloning, please view this page.

Reference:

1. GONTERO, Brigitte, María Luz CÁRDENAS, and Jacques RICARD. “A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle.” European journal of biochemistry 173.2 (1988): 437–443.
2. Bogdanove, Adam J., and Daniel F. Voytas. “TAL effectors: customizable proteins for DNA targeting.” Science 333.6051 (2011): 1843–1846.
3. !!参考往届iGEM的wiki不知道是否需要写上去，以及应该用什么样的格式
4. Deng, Dong, et al. “Structural basis for sequence-specific recognition of DNA by TAL effectors.” Science 335.6069 (2012): 720–723.
5. Pailler, J., W. Aucher, et al. (2012). “Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) of Escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane.” Journal of bacteriology 194(9): 2142–2151.
6. Schierle, C. F., M. Berkmen, et al. (2003). “The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.” Journal of bacteriology 185(19): 5706–5713.
7. Scholze, H. & Boch, J. TAL effectors are remote controls for gene activation. ‘‘Current Opinion in Microbiology’’ 14, 47–53 (2011).

8. Moscou, M. J. & Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effectors. ‘‘Science’’ 326, 1501–1501 (2009).
9. Conrado, R. J., G. C. Wu, et al. (2012). “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.” Nucleic acids research 40(4): 1879–1889.
10. Yang, Zhong, et al. “Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.” PloS one 6.7 (2011): e22981.