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Thermocycler Program: a gradient was used between 65°C and 80°C as annealing temperature. This time we used 30 cycles.

Afterwards we digested the products of the MutPCR with DpnI for 20min at 36°C and did a heat kill of the enzyme at 80°C for another 20min. After treating the PCR product with DpnI we did a Transformation into DH10B strain of E.coli using our standard protocol for transformation.

08/12/12

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ColonyPCR on the mammalian expression vector (MammoBrick) was done using our standard protocol for ColonyPCR. We picked 3 colonies. As primer we used CMV fwd (Tm: 58,8°C) and exPostRF rev (Tm: 59,6 °C). In the end we chose 60°C as annealing temperature.

08/13/12

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1.) MiniPrep of MammoBrick
2.) Digest of MammoBrick
3.) Digest of TAL-ORF
4.) Ligation of MammoBrick and TAL-ORF

1.) MiniPrep of the mammalian expression vector (MammoBrick) using Rothi Prep kit and its protocol
2.) We did a digest on the MammoBrick with EcoRI and SacII: enzymes will cut CMV, PostORF and PuroORF all together out of the rest of the vector (~1,5 kb band with gel-run).

Contents	[µl]
NEB Buffer 4	2
MammoBrick	5 ng
EcoRI-HF	0,5
SacII	0,5
ddH2O	end volume: 20 µl

We also digested MammoBrick using enzymes SpeI and PstI in order to use iGEM protocol for assembly of parts. We want to put the MammoBrick (now containing CMV, PostORF and PuroORF) together with our TAL-ORF (that later will contain our direpeats as an insert in order to have a complete TAL-Protein).

Contents	[µl]
MammoBrick	5 ng
PstI-HF	0,5
SpeI-HFF	0,5
BSA	0,2
ddH2O	end volume: 20 µl

3.) Digest of TAL-ORF with enzymes XbaI and PstI (see above)

Contents	[µl]
NEB Buffer 4	2
MammoBrick	5 ng
PstI-HF	0,5
XbaI-HF	0,5
BSA	0,2
ddH2O	end volume: 20 µl

4.) Ligation of MammoBrick and TAL-ORF

Ligation was executed and gel-run was carried out afterwards. Gel-run doesn’t show clear bands at defined positions. We decided to wait for results of sequencing.

08/16/12

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We had to amplify PostORF part again, using the following protocol:

Contents	[µl]
Phusion-Buffer HF	16
dNTPs	4
MgCl2	6,4
DMSO	4
Primer exPostORF fwd	4
Primer exPostORF rev	4
Phusion	1,6
ddH2O	8
Total	32

Total volume of 80 µl, used 4 Eppis á 25µl Thermocycler-Program same as for extensionPCR!

08/18/12

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1.) ColonyPCR on whole MammoBrick
2.) Repeating mutPCR of psB1C3 again
3.) New idea: provisory MammoBrick

1.) Colony PCR on whole MammoBrick construct , i.e. vector containing PostORF, PuroORF, CMV promotor and TAL-ORF, was carried out using our standard protocol for ColonyPCR. Unfortunately we couldn’t detect any clear bands on gel-run.

2.) Repeating mutPCR on psB1C3 again with same protocol from 08/11/12 again. Afterwards PCR-Purification using manufacturers protocol and then digest with DpnI to eliminate template, then transformation into DH10B strain of E. coli.

3.) New idea: producing as provisory MammoBrick In order to do so we did some research in Bioss’ ToolBox database and found a promising candidate to be used as our new MammoBrick. Only thing left to do is to clone our TAL-ORF into the new vector backbone, already containing everything we need.

Digest was prepared as follows:

Contents	[µl]
TAL-ORF [300 ng/µl]	3
BSA	5
NEB Buffer 4	5
XbaI	1
PstI	1
ddH2O	35

Contents	[µl]
provisory vector [200 ng/µl]	5
NEB Buffer 4	5
NheI	1
PstI	1
ddH2O	33

After digest was carried out in was applied on a agarose gel, where we were able to find clear definded bands. Bands were cut out and then purified using the manufacturers protocol for gel extraction. Afterwards Ligation was done as follows:br>

Contents	[µl]
TAL-ORF [7 ng/µl]	10
Vector [20 ng/µl]	2
Ligase T7	1
Ligase-Buffer	4
ddH2O	3

08/19/12

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1.) Transformation of provisory MammoBrick using our standard protocol for transformation.

08/20/12

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1.) ColonyPCR on provisory MammoBrick
After transformation of provisory MammoBrick into DH10B strain of E. coli today some colonies were picked and ColonyPCR was carried out.

We were not able to detect clear defined bands. Apparently cloning of provisory MammoBrick wasn’t successful and needs to be redone.

08/21/12

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extPCR in order to get CMV promotor out of psB1C3, as it is distributed in iGEM kit, was done again using already used protocol from... . It simply didn’t work this time either. Then we checked iGEM registry for further information and had to find out that provided sequencing data don’t containing any kind of CMV promotor as it was already described by iGEM another iGEM team in 2011. We always thought that distributed parts in kit were proofed, but know we know that it is not. So we have to find another plasmid on our own and design new primers in order to get another CMV promotor to put it in our MammoBrick. Current idea is to use CMV promotor present in our provisory MammoBrick.

08/24/12

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Repeating mutPCR all over again on psB1C3

We found out what kind of problem we had with all our previous mutPCRs on bsB1C3 vector backbone. Primer were able to bind at several loci throughout part between iGEM standard restriction sites. We did some research in registry of standard parts and found another part in backbone psB1C3 without described problems. We doesn’t care for the part, only for the backbone!

Then we repeated mutPCR using protocol of the several times before. Afterwards transformation into DH10B using our standard protocol.

09/05/12

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GGC reaction was done to get direpeats into provisory MammoBrick. Protocol was taken from Sanjana et al.

09/06/12

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1.) Transformation of GGC reaction product
2.) GGC reaction for effector
3.) PCR-Amplification of mutated backbone

1.)Transformation of GGC reaction product (see 09/05/12) into DH10B E. coli strain using our standard protocol for transformation. Plates containing Kanamycin!
2.)In addition GGC reaction to get an effector in provisory MammoBrick was carried out, too, as follows:

Contents	[µl]
BpiI	1
DTT [10mM]	1
Ligase T7	0,25
ATP	1
ddH2O	4,25
provisory Vector (100 ng)	0,75
Transcription Factor (9,9 ng) respectively (in another tube) FokI (30 ng)	1

Thermocycler program (step 1 and to 2 haven been repeated for 15 cycles):

Program for Thermocycler (15 cycles)
1.      37°C      5min
2.      20°C      5min
2.      4°C      //

3.) PCR-amplification of mutated backbone using protocol as follfows:

Contents	[µl]
circular, mutated plasmid	1
Primer fwd	2
Primer rev	2
Buffer HF	10
Phusion	0,5
DMSO	1,5
ddH2O	32 µl

After amplification PCR-purification was done using the manufacturers protocol and then digest was prepared overnight with EcoRI and PstI using iGEM standard protocols for assembly of standard parts.

09/07/12

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1.) Digest and Ligation of new PCR-extended and amplified CMV promotor into psB1C3 vector backbone provided by iGEM registry in order to send it in as improved part for broken CMV promotor that is actually distributed by the registry.

09/08/12

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Trying to get AID into provisory MammoBrick. Therefore we did a GGC reaction using protocol taken from Sanjana et al.
Then we did ColonyPCR using our elaborated standard protocol for ColonyPCR.

We also did ColonyPCR with same protocol for transcription factor.

Later that day we repeated GGC reaction in order to produce MammoBrick all over again as follows:

Contents	[µl]
BsaI	1,5
NEB Buffer 4	1
BSA	1
ATP	1
T7-Ligase	0,25
Cut TALEN [80 ng]	1
postORF [23 ng]	1
CMV [27 ng]	3
Puro [22 ng]	2

09/11/12

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1.) GGC to get direpeats into TAL-TF
2.) GGC to get direpeats into AID
3.) Transformation of above products into DH10B competent cells

09/16/12

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Today we decided to use another protocol for producing ready to use TALEs. Until today we always used GGC protocol following Sanjana et al., but now we want to try it using protocol following Morbitzer et al. In comparison to the Sanjana-paper we are now using only 30 U/µl enzyme activity (before 3000 U/µl). Doing this we also had to adjust reaction time in thermocycler from 3h to 9h reaction time with more cycles. After this was accomplished we transformed it into ccdb-resistant bacteria following our elaborated standard transformation protocol and waited for the next day to hopefully pick some colonies.

09/17/12

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GGC reaction done on the day before looks like a great success. We have lots of colonies this time. We picked some early that day and did miniprep on them later that day so that they can be sequenced.
After this success (on first look) we decided to repeat MammoBrick GGC using the same protocol by Morbitzer et al.

09/18/12

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Sequencing is positive and our samples look as they should look like! It seems that we are finally able to produce TALEs á la freiGEM using direpeats still in psb1C3 vector backbone.
Now we have to combine it at least the transcription factor into it, but also the AID in order to help Potsdam!

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