Difference between revisions of "Part:BBa K747099"

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	Eukaryotic TALE expression vector:</b><br><br>		Eukaryotic TALE expression vector:</b><br><br>
−	Once the MammoBrick was ready, we inserted the TAL open reading frame and thereby evaluated, how easy it would be for future iGEM students to expression any desired ORF in eukaryotic cells.<br><br>	+	Once the [[BBa K747099]] was ready, we inserted the TAL open reading frame and thereby evaluated, how easy it would be for future iGEM students to expression any desired ORF in eukaryotic cells.<br><br>
	Designing the TAL open reading frame:<br>		Designing the TAL open reading frame:<br>

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Revision as of 21:01, 28 September 2012

Eukaryotic TAL expression plasmid

Eukaryotic TALE expression vector:

Once the [[BBa K747099]] was ready, we inserted the TAL open reading frame and thereby evaluated, how easy it would be for future iGEM students to expression any desired ORF in eukaryotic cells.

Designing the TAL open reading frame:
For this purpose, we designed a TAL ORF by adding the following modifications to the TAL open reading frame in pTALEN (v2) NG:

1. We removed all EcoRI, XbaI, SpeI, PstI, BsmBI, BbsI and PmeI restriction sites.
2. We replaced the BsaI restriction sites for inserting direpeats by BsmBI sites, because – according to the manufacturer - BsmBI is better suited for digest over one hour.
3. We added a consensus RBS in front of the ORF for expression in bacteria
4. We added a His-Tag to the n-terminal end to allow protein purification.
5. We flanked the whole sequence with the iGEM prefix and suffix.
6. Most importantly, we replaced the FokI nuclease at the C-terminal end of the protein by one of our inventions: The Plug and Play Effector Cassette. This whole construct was synthesized by IDT.

Plug and Play Effecor Cassette: Our project was designed to enable future iGEM teams to easily use the powerful TALE technology. On top of that, we wanted to built a TALE platform which allows iGEM students to freely develop their own TAL constructs. We therefore invented the easy-to-use Plug and Play Effector Cassette (PPEC), which can be used to fuse BioBricks, that are in the Golden Gate standard (hyperlink zu Golden Gate Cloning), to the c-terminus of the TAL protein. The PPEC



consists of two BbsI binding sites that point in opposite directions. Digestion with BbsI leads to removal of the PPEC and to the formation of sticky ends at which the upstream sticky end (GGCA) are the last 4 nucleotides of the tal protein and the downstream sticky end (TAAA) contains the stop codon. When an equimolar amount of the effector containing plasmid (flanked also by BbsI sites and the same overlaps) is added to the GGC mix, the effector is cut out of the iGEM vector and ligated into the eukaryotic TAL expression vector in-frame and without a scar. We have optimized this reaction by systematically testing different reaction buffers and thermocycler programs and came up with the following protocol:

Component	Amount (μl)
BpiI/BbsI (15 U)	0,75
T4 Ligase (400 U)	1
DTT (10 mM)	1
ATP (10 mM)	11,5
G-Buffer (10x, Fermentas)	1
parts	40 fmoles each
ddH2O	Fill up to 10
Total	10

Thermocycler programm:
1.     37°C, 5 min
2.     20 °C, 5 min
go back to 1. 20 times
4.     50°C, 10 min
5.     80°C, 10 min
5.     4°C, ∞

But even Golden Gate cloning is not 100 % efficient. In order to remove those plasmids that did not take up a vector insert, we added the restriction site of the blunt end cutter PmeI (MssI) to the PPEC. We chose PmeI because it has a 8 bp binding site, which is very unlikely to occur in the gene of an effector that you would like to fuse with the TAL gene. So after performing the Golden Gate reaction described above, we digested with MssI fast digest (fermentas) according to the following protocol.

Component	Amount (μl)
GGC-Product	10
PmeI/MssI FastDigest	1
Fast Digest Buffer (10x)	1,5
ddH2O	2,5
Total	15

Thermocycler programm:
1.     37°C, 1h
2.     80 °C, 20 min
5.     4°C, ∞

This linearizes all vectors that do not contain the effector (at least, we do not see colonies on the negative control plate). To be sure, these linearized vectors do not religate, perform the following digest with T5 exonuclease, which specifically removes linearized DNA:

Component	Amount (μl)
Product of PmeI digest	7,5
T5 Exonuclease	1
Total	8,5

Thermocycler programm:
1.     37°C, 1h
2.     80 °C, 20 min
5.     4°C, ∞

Sequence and Features