Team:Colombia Uniandes/NickoJournal

Dear Journal! :)

Here we will place all the information about the work we have been doing during this time, it will be named by date. We hope you enjoy our work and thoughts as much as we do!

Our Notebook

18-Jun-2013

Hello! Hello! Hola! Hola! After we went over the project, we proceed to design our primers so we could place our order, taking into account that we are going to use Fusion PCR as our main protocol. If you want to check our primers, go to How to: Parts. Our primers take around 2 weeks to arrive… sooo meanwhilee all the team started making our E.coli babies electrocompetent, so we can work with them later on.

25-Jun-2013

So today we took our bacterial cells (Top 10) onto an LB medium (no antibiotics). We let it ON at 37 °C on the shaker. Also we prepared all the material we need for tomorrow: ddwater, 10 % glycerol, LB medium and microfuge tubes.

26-Jun-2013

Today we are going to start preparing our cells, we inoculated in the morning and placed all our material that needs to be cold in the fridge 4 °C(ddWater and glycerol), also the centifuge rotor needs to be chilled. Then we just waited for the perfect OD and made our cells around 4:00pm. We made a lot!

We placed them in the -80, ready to start our work!!!

2-Jul-2013

Our primers are FINALLY here and we are anxious to start working!!! So we made our plan(in theory)on big steps for the next weeks!:

1. DNA extraction from E.coli and Cupriavidus metallidurans CH34

2. PCR

 2.1 PrcnR, RBS, rcnR, stop (Amplified as one piece)
 2.2 PrcnA,RBS
 2.3 hoxN, stop

3. Fusion PCR

 3.1 PrcnA/hoxN
 3.2 PrcnR,RBS,rcnR/PrcnA/hoxN

On each PCR add 30 steps more of making gels for confirmation, again and again and again.

3-Jul-2013

Fortunately our team member Silvia Cañas already had a DNA extraction from E.coli so she donated it to us so we could start working! Thank you Silvia! :) We asked for Cupriavidus metallidurans to our teacher Jenny Dussan at her lab here in the university. She is giving us the strain tomorrow morning! Thank you Jenny! This done, did the extraction of C.metallidurans and store it in the -30 °C.

4-Jul-2013

Today we started with the 2-step PCR of PrcnR, RBS, rcnR, stop (Fragment that will be called "R" from now on). Results were negative :(

5-Jul-2013

Today we repeated "R" PCR and also did hoxN with the Cupriavidus metallidurans genome. The quantities used for the reaction of hoxN are shown in the table below. Results were negative :( as we could see in our sad, SAD gel.

9-Jul-2013

So, our PCRs are NOT working....We think our problem is the length of the primers and their constitution that could form secondary structures very VERY easily. That's why we tried a new procotol of PCR for "R" with a temperature gradient, and we tried new buffers for hoxN. Reagents and quantities are shown in the table below.

We got a POSITIVE result on hoxN, and POSITIVE with "R" at "low" temperatures of annealing. Here is our BEAUTIFULLL gel.

GoodNewsGEL

On 5,7,8,9 we can see "R" fragment of 359bp. On 12 we can see hoxN fragment of 837bp.

11-Jul-2013

Today we did the PrcnA PCR and it didn't work out.

15-Jul-2013

We started hoxN PCR with the phusion primers, over the C. metallidurans genome, using 2-step PCR protocol.

Results are negative.

17-Jul-2013

We repeated hoxN PCR with the fusion primers over the genome and PrcnA PCR with E.coli genome.

Nothing worked OUT :( !!!

We are going to try to do the PCR of hoxN with the phusion primers over the fragment already amplified and see, as fas as PrcnA goes...we will try a temperature ramp with a 3-step PCR protocol. End for today :(

18-Jul-2013

Today we did E. coli chimiocompetent cells to mRFP transformation using TransforrmAid Bacterial Transformation Kit (Thermo Scientific). We did mRFP transformation from iGEM plate.

19-Jul-2013

We have transformants!!!! Beautiful, aren´t they?

Transformants

24-Jul-2013

Today we are going to do pRcnA PCR. Besides iGEM kit, we used Phusion High Fidelity PCR kit and tested its two buffers: Phusion HF reaction Buffer and Phusion GC Reaction Buffer. We used a 2-step PCR protocol. Reagents and quantities of iGEM kit are the same we have been using until now, but reagents and quantities used for Phusion High Fidelity PCR kit are shown in the table below.

25-Jul-2013

We did hoxN PCR by using the same kits than yesterday and 2-steps protocol. However, results are negative :'(.

26-Jul-2013

Since we have been having troubles with PCRs, we decided to do genome extraction of our bacteria again. For this purpose we used Easy DNA Isolation Kit, Invitrogen. We followed manufacturer's instructions. We confirmed the extraction by gel electrophoresis. Genomes of C. metallidurans and new E.coli Extracted :D

GenomeNewExtractionWithOUTRnase

• Remember you can review protocols in the section named Protocols

29-Jul-2013

Today we did PCR of hoxN from the new C. metallidurans genome exracted and guess what? We got hoxN a beatiful band around 600 pb :D :D. Blue arrow shows HoxN and yellow arrow shows rcnR...FAIL

GelHoxN

30-Jul-2013

We did PCR of rcnR using the E. coli genome extracted last Friday and Phusion High Fidelity PCR kit. On the gel we got a diffuse band around 400 pb, so we could not conclude anything.

31-Jul-2013

We did PCR of pRcnA and rcnR again using a annealing temperature gradient between 45°C and 60° and using the two buffers: Phusion HF reaction Buffer and Phusion GC Reaction Buffer. On the gel we saw a band that corresponded to pRcnA when the annealing temperature had been around 53°C and GC buffer had been used. On the other hand, in rcnR reactions we got a band when buffer HF had been used and when annealing temperature was around 58°C. So...u CAN BARELY see them BUT THEY ARE THERE!

Prcna,rcnR

01-Aug-2013

Today we did hoxN-rfp phusion. We used Phusion High Fidelity PCR kit, Phusion HF reaction Buffer and the amounts of other reagents described below. We got our first phusion!!! What a great day :)

hoxNRFP fusion!

02-Aug-2013

Another try of PrcnR-rcnR and then again...nothing.

rcnR

05-Aug-2013

Since we obtained PrcnA and HoxN, we made a Prcna-HoxN fusion and we also amplified hoxN-RFP fusion. All was obtained and we can see that in our beautifull gel :)Go guys!! The fisrt column next to the DNA ladder is HoxN-RFP fusion on the first pink arrow, on the second pink arrow there is our fusion Prcna-HoxN.

Prcna-HoxN and HoxN-RFP

07-Aug-2013

Today we decided to digest our parts with EcoRI in order to insert our parts in the backbone. Thats why first we did the digestion with EcoRI on the Backbone and also the same process with the parts. We digested for 2 hours and then desactivated the ennzymes at 80°C for 20 min. For protocol see: https://www.neb.com/products/r0101-ecori#tabselect2

We then used antarctic phosphatase for the backbone for 1h. For protocol see: https://www.neb.com/protocols/1/01/01/vector-dephosphorylation-protocol

Finally we let the ligation all night at room temperature.

09-Aug-2013

Today we desactivated the ligase at 80°C for 15 min and started THE TRANSFORMATION!

We transformated our cells and let them in 37°C. Lets see tomorrow :)

10-Aug-2013

They were NO colonies At ALL! not even halfffffff, not even a tiny oneee!!

We are repeating process on monday.

13-Aug-2013

Today we did electrocompetent cells with E.coli DH10B and we tranformed and well...hopefully we will see something tomorrow!

Hopes up!

14-Aug-2013

We have some colonies! but only on HoxN-RFP fusion! We are going to leave those on ON and we are transforming Prcna-HoxN again.

15-Aug-2013

Transformation not succesfull, do it one more time!

16-Aug-2013

Transformation not succesfull, do it one more time!

19-Aug-2013

Transformation not succesfull, do it one more time!

20-Aug-2013

Tranformation succesfull!!!! :) Growing on ON!

21-Aug-2013

Miniprep DAY! :)

Parts Ready!

23-Aug-2013

We are sending Prcna-HoxN and HoxN-RFP :) We are still trying to get Prcnr-rcnR so lets go!

24-Aug-2013

We tried PCR Prcnr-rcnR and IS NOT THEREEEEEEEEEEEE!