Difference between revisions of "Team:Paris Bettencourt/Notebook/Enzyme"

Weeks 8-14th and 15-21th August

On August the 11th our gBlocks finally arrived, so we could really start our work on the enzyme studies.
The first step was to clone the vector in which we will clone all of our enzymes. The vector (pCOLA-FS) is based on the pCOLA plasmid, only it only contains one T7 promoter, no cloning sites incompatible with the PhytoBricks, and also contains the lac operon under the remaining T7 promoter.
The idea of cloning the lac operon is to simplify the screening for the rightly cloned plasmids.

Gibson Assembly of the pCOLA-FS plasmid

The pCOLA-FS plasmid was assembled by Gibson Assembly from the following fragments:

• pCOLA-FS synthetic fragment This fragment is 1925bp long.
• pCOLA PCR product, amplified from primers FNS_15 and FNS_16. This fragment is 2128bp long.

Golden Gate of the various pCOLA-FS plasmids

After we obtained the pCOLA-FS plasmid and checked it by sequencing, we began the cloning of the different pCOLA-FS plasmids carrying the different enzymes for testing.
For doing that, the first step was getting the fragments to clone using BsaI cloning.

The PCR reaction was carried out using ThermoFischer Phusion polymerase, because of its proofreading activity, and the PCR was carried out in a volume of 50uL (10uL 5X HF Buffer, 1uL 10mM dNTPs, 2.5uL of each primer, 2uL of the template (for 20bg), 1,5uL DMSO, 0.5uL pol and 30uL water). The PCR cycle was as follows: 98ºC 30’ – (98ºC 10’’ – 60ºC 20’’ – 72ºC 1’30’’)x33 – 72ºC 10’

After purification we run a gel to confirm the amplification:

Wells: 1.CBD – 2.pbuI_Ph – 3. bpuI – 4.tanLpI_ph – 5.tanLpI – 6.BG1_Ph – 7.BG1 – 8.xylE_Ph – 9.xylE – 10.PPO2_Ph – 11.PPO2 – 12.catA_Ph – 13.catA

Weeks 22-28th and 29th August – 4th September

During these two weeks we performed the Golden Gate to get our several plasmids.
It took us longer than we had originally expected because we run into some problems regarding the construction of the plasmids.
Luckily, we were able to figure the mistakes out. There were no mistakes in our designing of the cloning (uffff, we are happy about that, it would have been a disaster). There were nonetheless mistakes in the way we performed the protocol for Golden Gate.

We used the NEB Golden Gate Mix throughout all the attempts.

We started first by amplifying the fragments for the Golden Gate (as said already in the past week). IDT says that PCR should not be performed on their GBlocks in the case of them being bigger than 1000pb, which in all of our cases they are. Nonetheless, we needed to perform the PCR on the fragments because we needed different tails added to them.

The first Golden Gate protocol that we did was the one that is proposed by NEB for Golden Gates with up to 5 fragments (which is our case).
Total volume of reaction is 20uL. We added 2uL of the NEB Golden Gate Buffer, 75ng of plasmid (in our case 1.39uL), we kept a 2:1 molar ratio for each of the inserts, added 1uL of the Golden Gate Assembly Mix and added water up to the final volume.
We performed the proposed protocol of 1h at 37ºC an 5 min at 55ºC, and transformed the results into E. coli DH5alfa.
The following day we had a high number of colonies in our plates, all of them white (correct colour for the blue/white screening that we are performing).

Nonetheless, we performed PCR with our checking primers (ch1 and ch2/ch8) and all our bands were smaller than they were supposed to be. They seemed to be the size of what would be in the plasmid if the lacZ fragment had been correctly excised, and something quite small (up to 300pb) had been inserted.

The PCR was with Dreamtaq polymerase and as follows:
Volume per reaction 10ul:
- Mastermix --> 5ul
- ch1 --> 0.5 ul
- ch8 --> 0.5 ul
- DNA* --> 4ul
* DNA is actually the selected colony diluted in 20ul of destiled water.

PCR's settings:
1st- 10min -- 98C --> Cell lysis
2nd- 3min -- 95C --> Activation
3th- 30sec -- 95C --> Denaturailzation
4th- 30sec -- 55C --> Hybridation
5th- 2min -- 72C --> Elongation
Repeat from 3th to 5th 25 times
7 minutes -- 72C --> Final elongation

After the PCR we run an electrophoresis with the product:

The settings for the Golden Gate reaction were as follows:
1. 37 C - 5 min
2. 37 C - 2 min
3. 16 C - 5 min
4. 50X back to 2
5. 37 C - 5 min (final digest)
6. 50 C - 10 min
7. 80 C - 10 min

The Golden Gate product was transformed by heat shock into thermo competents E.coli DH5-alpha and they were growth in LB plates with Kanamycin (50ug/ml), X-gal (20ng/ml) and IPTG (0.1mM), and growth overnigth at 37C.
We did so to do Blue/White screenning.
(Following the protocoles in the protocole section). We also transformed a negative control with the intact plasmid without being processed by the GG assembly.

Next day we observed white and blue colonies in the plates, and all blue in the negative control as is was expected. Therefore we selected those colonies of each clone which were white and we did colony PCR on them.
The PCR was performed with Dreamtaq polymerase and as follows:
Primers ch1 and ch8.
Volume per reaction 25ul:
- Mastermix --> 12.5ul
- Water --> 5ul
- ch1 --> 2.5 ul
- ch8 --> 2.5 ul
- DNA* --> 2.5ul
* DNA is actually the selected colony diluted in 20ul of destiled water.
PCR's settings: 1st- 10min -- 98C --> Cell lysis
2nd- 3min -- 95C --> Activation
3th- 30sec -- 95C --> Denaturailzation
4th- 30sec -- 55C --> Hybridation
5th- 2min -- 72C --> Elongation
Repeat from 3th to 5th 25 times
7 minutes -- 72C --> Final elongation

After the PCR we ran an electrophoresis with the product but the gel was empty, it might have been because the polymerase Dream taq we were using was spoilt since the control wasn't amplifyed, therefore we repeated the PCR with another aliquote of Dream taq polymerase under the same conditions of PCR and after we run a new electrophoresis.

The Control plasmids is a PCR where only clean plasmid (after miniprep) was added.
The Control clony corresponds with a common colony PCR with an strain which contains the pFNS0, our backbone plasmid.
Finally we had reason to be happy :D. Since we had some positives. In this case all the clones except BGI.
Therefore we could start doing the phusion proteins, since we had all the clones for the proteins with only the His-tag.

In parallel we run another PCR but changing a detail, we realized that if we did the cell lysis in the thermocycler, as it had been done until now (see above), the resolution of the electrophoresis was not so good, then we decided to repeat the electrophoresis but doing the lysis before in a thermoblock.
The protocol followed is the following:
1º 5min - 100C --> Cell lysis
2º 5sec - vortex --> Homogenization
3º 3min - centrifuge 6000g --> Pelleting the cell debris
4º collect SuperNatant

The PCR was with Dreamtaq polymerase and as follows:
Primers ch1 and ch2.
Volume per reactio 50ul: (50ul to ensure that we have DNA in the 2ul of DNA we use)
- Mastermix --> 25ul
- Water --> 19ul
- ch1 --> 2 ul
- ch2 --> 2 ul
- DNA* --> 2 ul
*It is the SN collected after the cell lysis.

PCR's settings:
1nd- 3min -- 95C --> Activation
2th- 30sec -- 95C --> Denaturailzation
3th- 30sec -- 55C --> Hybridation
4th- 2min -- 72C --> Elongation
Repeat from 2th to 5th 25 times
7 minutes -- 72C --> Final elongation

After this an electrophoresis (agarose 1%) was carried out:

As we can see this gel shows again positives , even one for BGI. We decided to do miniprep of the following clones:
1.3 and 1.4 (bpuI)
2.3 and 2.5 (catA)
3.3 and 3.4 (tanLpI)
4.4 and 4.5 (PPO)
5.3 (BGI)

The results of the miniprep of this clones are:

After the normal proteins were golden gated, we starte the GG for the six fuision proteins. To do that we did PCR first.
This GG mix contains the gblocks amplified two weeks ago with the primers which allow to have cohesive end in 3' with the 5' of the CBD protein, once it has been digested with BsaI, and the CBD gblock we used is the one coming directly from the product IDT supplied us.
The protocol we followed was the same that worked before:

For a final volume of 10 uL
x 40 fmol of vector and insert
1ul Buffer 10x
0.5ul Assembly mix
x dH2O (until 10ul)
-----------------------------
10 uL total

Settings
1. 37 C - 5 min
2. 37 C - 2 min
3. 16 C - 5 min
4. 50X back to 2
5. 37 C - 5 min (final digest)
6. 50 C - 10 min
7. 80 C - 10 min

To compute the quantities to add of DNA we used the excel sheet shown before.