Team:UANL Mty-Mexico/Notebook

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Notebook

Reference Data

Part Name	Part Short name	In pSB 1A3 (bp)	In pSB 1C3 (bp)	In pUC57 (bp)	Part size (bp)
RBS + TetR	TetR	2,925	2,802	3,478	732
pLac + GFP	GFP	3,162	3,034	3,715	964
pCons LacI	LacI	3,483	3,352	4,036	1,282
pTetR + mCherry	mCherry	3,107	2,957	3,660	887
BBa_K1140005	3-E1	2,811	---	---	935

This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "RBS + TetR". Another important aspect is that we were working with the plasmids pSB 1C3, pSB 1A3 and pUC57 so here there is the information about the length of the part and the length in each plasmid.

August 4th, 2013

Today we prepared solutions in order to start working on the lab the following day. The solutions will be required for the MiniPrep, Transformation. We also prepared aliquots, mQ water with RNAse, etc.

August 5th, 2013

Today we started with the transformations of the synthetic DNAs that just arrived.

August 6th, 2013

We inoculated the colonies obtained from the transformations.

August 7th, 2013

This day we did Minipreparation of DNA. The parts that were obtained were the following: LacI, GFP, mCherry, TetR and 3-1E

August 8th, 2013

Test of mCherry with Thermo-mixer-Cualitative experiment

20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red colour after several hours at 42ᵒC. The experiment was performed following these parameters.

	Start	End	Hours
37ºC	2:50 pm	7:02 pm	4 hr 12 min
42ºC	7:05 pm	10:35 am	15 hr 40 min

Most tubes shown development seeing them on the light but there was no color shown.

August 10th, 2013

20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone), following these parameters:

Temperature: 37ᵒC

Revolutions: 900 rpm

Start: 11:35 am -> 9:00 am (Time 21:35 hours)

The tubes show mCherry expression, some more intense than others. The experiment was repeated at 30°C overnight with a volume of 500 µL at 30°C with tubes of 1.5 ml. They were left at 4°C meanwhile they were incubated.

Set	Temperature	Velocity	Time	Live
Black Set	42ºC	900 rpm	15 hours	:(
Blue Set	37ºC	900 rpm	21 hours	:)
Red Set	32ºC	900 rpm		:)

Time 10:25 – 9:15 = 22:50 hrs

August 12th, 2013

The experiment was repeated at 42°C allowing the oxygen in to the tube. It began at 2:15 pm and ended at 1:20 pm of the next day.

Figure 1. Tubes with the mCherry pellet that were incubated at 42ºC.

August 16th, 2013

Electrophoresis gel of the digestions from 15/08/13

DNA	1x	5x
DNA	2 µL
EcoRI	0.3 µL	1.5 µL
PstI	0.3 µL	1.5 µL
Buffer O (Fermentas)	1 µL	5 µL
	10 µL	50 µL

August 18th, 2013

There where no colonies present from the previous day.

Ligation with a 1:1 ratio DNA:Vector. Is was performed with the following parameters:

	Size (bp)	Vector	DNA:Vector
pTetR	768	2.8	0.33/1
pCons-LacI	1326	1.6	0.62/1
pTetR-mCherry	960	2.2	0.45/1
pLacI-GFP	1005	2.1	0.47/1

August 19th, 2013

This ligation was not successful in the transformation.

Dishes of:pTet-mCherry, pCons-LacI, pTetR, pLacI-GFP, and the 3-1E DNA were planted again.

August 26th, 2013

All the dishes were transformed but only the pLacGFP grew successfully.

August 28th, 2013

Today we picked up colonies from the dishes from the previous day. We also prepared new competent cells.

August 29th, 2013

All the dishes were successfully transformed and we planted in petri dishes. We got colonies of all the parts, they grew in the test tubes and miniPrep was done.

August 30th, 2013

The MiniPrep was successful and we did digestions with EcoRI. Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. *They were planted in Ampicilin dish.

Figure 3. Electrophoresis gel from the August 30 miniPreps

August 31th, 2013

MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.

Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP

*They were planted in Ampicilin dish.

September 11th, 2013

Digestions with EcoRI and PstI and Ligations

September 12th, 2013

We didn't obtained any part so we started again transforming them one more time.

September 13th, 2013

There were no results presented so we transformed again.

September 14th, 2013

We got no results in the construction desired.

September 19th, 2013

Quantification with the Nanodrop of the previous samples

	ng/µL	260/280
pSB1C3	1293.4	1.99
GFP	133.5	1.84
TetR	119.6	1.78
tRFP	104.3	1.74
pConsLac	130.5	1.59

September 20th, 2013

We prepared new calcium competent cells, E. coli strain Top10.

September 23th, 2013

We did the transformations again for all the parts.

September 24th, 2013

Again there where no results.

Note: The constructions with the pSB1A3 and pSB1C3 showed no results but the controls with pUC vector were fine and did work in our experiments.

October 9th, 2013

New Digestions with EcoRI and PstI.

October 10th, 2013

New ligations were done

October 11th, 2013

New Petri dishes were made, and use for transformation today.

October 13th, 2013

We inoculated the colonies obtained from the transformations.

October 14th, 2013

We did minipreparation of DNA. After, the DNA was digested with EcoRI, PstI and HinfI for an enzymatic analysis (our Gel #700!!!). After hours, we did a electrophoresis and we realised that 2 new parts were now in the pSB 1C3 vector.

Figure 4. Electrophoresis gel from the August 30 miniPreps cut with *Hin*fI, Lambda-*Pst*I as marker.

October 17th, 2013

We started a new test in order to measure number of plasmids per cell in M1, M2 and M12.

October 18th, 2013

First results for number of plasmids.

October 21th, 2013

More results for number of plasmids.

October 22th, 2013

A test for measuring expression of GFP (in pUC57 and pSB1C3) was prepared in microcentrifuge tubes with LB medium and IPTG 1mM for 17 hours at 37 °C (in bacteria E. coli Top10) in thermomixer.

We still have problems with expression of mCherry, so we did new cultures with stored (red) M1, M2 and M12 in order.

October 23th, 2013

We used fluorometer to measure expression of GFP, but any fluorescence was detected, so new microcentrifuge tubes with GFP construction were prepared at same conditions of last day.

The cultures of M1, M2, M11 and M12 were red and presented fluorescence, so we did minipreparation and culture again them in new tubes. With the DNA that was obtained, a digestion with EcoRI and PstI was done and a new ligation.

October 24th, 2013

Again, any fluorescence was detected for GFP cultures. New microcentrifuge tubes with GFP construction were prepared with IPTG 0.5 mM.

A new transformation was done, using new mCherry and latest TetR and LacI ligations.

Cultures of M11 and M12 grew, but any with M1 and M2. We did minipreparation and the DNA was measured in nanodrop.

October 25th, 2013

Residual fluorescence was detected for GFP cultures.

We inoculated the colonies obtained from the transformations.

October 26th, 2013

Cultures of mCherry grew, but the others did not. We did minipreparation of that tubes and digested them with EcoRI, PstI and HinfI. The results can be seen on gel # 709 (EcoRI/PstI) and 710 (HinfI). Probably, we have multiple clones with mCherry in pSB1C3.

New ligations of TetR, LacI and GFP with pSB1C3 were done and a transformation was made in a new competent stock, Top10 strain.

Figure 5. Electrophoresis gel (709) with the *Eco*RI/*Pst*I clones digestion, Lambda-*Pst*I as marker.

Figure 6. Electrophoresis gel (710) with the *Hin*fI clones digestion, Lambda-*Pst*I as marker.

October 28th, 2013

We did minipreparation and digested them with HinfI to identify more positive clones. As a result we get at the most two positive clones, one for pSB-mCherry and one for pSB-GFP

Figure 7. Electrophoresis gel with the *Hin*fI clones digestion for mCherry, GFP, LacI TetR with pSB1C3 vector as control, Lambda-*Pst*I as marker.

Stability

For the stability experiments the tests were performed from September 14 to 20. The protocols are in the section of Safety. Click here

For the fluorescence experiments the tests were performed from September 10 to 27. The protocols are in the section of Wetlab. Click here

@@ Line 148: / Line 148: @@
 <center>
 <figure>
-<img src="https://static.igem.org/mediawiki/2013/9/99/37Cunal2013.jpg" width=500px style="border:1px solid black" >
+<img src="https://static.igem.org/mediawiki/2013/9/99/37Cunal2013.jpg" width=400px style="border:1px solid black" >
 <figcaption><span class="text-muted"><br>Figure 1. Tubes with the mCherry pellet that were incubated at 42ºC.</span> <br>
 </figcaption>
@@ Line 158: / Line 158: @@
 <center>
 <figure>
-<img src="https://static.igem.org/mediawiki/2013/2/2b/Fig2_657.png" width=400px style="border:1px solid black" >
+<img src="https://static.igem.org/mediawiki/2013/2/2b/Fig2_657.png" width=300px style="border:1px solid black" >
 <figcaption><span class="text-muted"><br>Figure 2. Electrophoresis gel of the digestions from 15/08/13</span> <br>
 </figcaption>
@@ Line 217: / Line 217: @@
 </tr>
 <tr>
-<td>pCons-Lac1</td>
+<td>pCons-LacI</td>
 <td> 1326</td>
 <td>1.6</td>
@@ Line 228: / Line 228: @@
 <td>0.45/1</td>
 </tr><tr>
-<td>pLac1GFP</td>
+<td>pLacI-GFP</td>
 <td> 1005</td>
 <td>2.1</td>
@@ Line 252: / Line 252: @@
 <center>
 <figure>
-<img src="https://static.igem.org/mediawiki/2013/0/07/662b.jpg" width=500px style="border:1px solid black" >
+<img src="https://static.igem.org/mediawiki/2013/6/6e/MinPreps30Aug.png" width=300px style="border:1px solid black" >
 <figcaption><span class="text-muted"><br>Figure 3. Electrophoresis gel from the August 30 miniPreps</span> <br>
 </figcaption>
@@ Line 282: / Line 282: @@
 <tr>
 <th><b></b></th>
-<th><b>Ng/µL</b></th>
+<th><b>ng/µL</b></th>
 <th><b>260/280</b></th>
 </tr>
@@ Line 314: / Line 314: @@
 <p><b>September 20th, 2013</b></p>
-<p align="justify">We prepared new calcium competent cells. </p>
+<p align="justify">We prepared new calcium competent cells, <i>E</i>. <i>coli</i> strain Top10. </p>
 <br>
 <p><b>September 23th, 2013</b></p>
@@ Line 334: / Line 334: @@
 <br>
 <p><b>October 11th, 2013</b></p>
-<p align="justify">New dishes were transformed and plated.</p>
+<p align="justify">New Petri dishes were made, and use for transformation today.</p>
 <br>
 <p><b>October 13th, 2013</b></p>
@@ Line 344: / Line 344: @@
 <figure>
 <center>
-<img src="https://static.igem.org/mediawiki/2013/7/76/700Zorrab.png" width=500px style="border:1px solid black" >
+<img src="https://static.igem.org/mediawiki/2013/7/76/700Zorrab.png" width=300px style="border:1px solid black" >
-<figcaption><span class="text-muted"><br>Figure 4. Electrophoresis gel from the August 30 miniPreps</span> <br>
+<figcaption><span class="text-muted"><br>Figure 4. Electrophoresis gel from the August 30 miniPreps cut with <i>Hin</i>fI, Lambda-<i>Pst</i>I as marker.</span> <br>
 </figure>
 </center>
@@ Line 369: / Line 369: @@
 <br>
 <p><b>October 24th, 2013</b></p>
-<p align="justify">Again, any fluorescence was detected for GFP cultures. New eppendorf tubes with GFP construction were prepared with IPTG 0.5 mM.</p>
+<p align="justify">Again, any fluorescence was detected for GFP cultures. New microcentrifuge tubes with GFP construction were prepared with IPTG 0.5 mM.</p>
 <p>A new transformation was done, using new mCherry and latest TetR and LacI ligations.</p>
 <p>Cultures of M11 and M12 grew, but any with M1 and M2. We did minipreparation and the DNA was measured in nanodrop.
@@ Line 375: / Line 375: @@
 <br>
 <p><b>October 25th, 2013</b></p>
-<p align="justify">Any fluorescence was detected for GFP cultures.</p>
+<p align="justify">Residual fluorescence was detected for GFP cultures.</p>
 <p>We inoculated the colonies obtained from the transformations.
 </p>
 <br>
 <p><b>October 26th, 2013</b></p>
-<p align="justify">Cultures of mCherry grew, but the others didn´t. We did minipreparation of that tubes and digested them with <i>Eco</i>RI, <i>Pst</i>I and <i>Hin</i>fI. The results can be seen on gel # 708 and 709. Probably, we have multiple cultures with mCherry-pSB1C3.</p>
+<p align="justify">Cultures of mCherry grew, but the others did not. We did minipreparation of that tubes and digested them with <i>Eco</i>RI, <i>Pst</i>I and <i>Hin</i>fI. The results can be seen on gel # 709 (<i>Eco</i>RI/<i>Pst</i>I) and 710 (<i>Hin</i>fI). Probably, we have multiple clones with mCherry in pSB1C3.</p>
-<p>New ligations of TetR, LacI and GFP with pSB 1C3 were done and a transformation was made in a new competent stock, Top10 strain.
+<p>New ligations of TetR, LacI and GFP with pSB1C3 were done and a transformation was made in a new competent stock, Top10 strain.
+</p>
+<br>
+<figure>
+<center>
+<img src="https://static.igem.org/mediawiki/2013/2/2d/Gel709a.jpg" width=250px style="border:1px solid black" >
+<figcaption><span class="text-muted"><br>Figure 5. Electrophoresis gel (709) with the <i>Eco</i>RI/<i>Pst</i>I clones digestion, Lambda-<i>Pst</i>I as marker.</span> <br>
+</figure>
+</center>
+</p>
+<br>
+<br>
+<figure>
+<center>
+<img src="https://static.igem.org/mediawiki/2013/5/59/Gel710a.jpg" width=500px style="border:1px solid black" >
+<figcaption><span class="text-muted"><br>Figure 6. Electrophoresis gel (710) with the <i>Hin</i>fI clones digestion, Lambda-<i>Pst</i>I as marker.</span> <br>
+</figure>
+</center>
+</p>
+<br>
+<p><b>October 28th, 2013</b></p>
+<p align="justify">We did minipreparation and digested them with <i>Hin</i>fI to identify more positive clones. As a result we get at the most two positive clones, one for pSB-mCherry and one for pSB-GFP
+</p>
+<br>
+<figure>
+<center>
+<img src="https://static.igem.org/mediawiki/2013/0/0a/713.jpg" width=500px style="border:1px solid black" >
+<figcaption><span class="text-muted"><br>Figure 7. Electrophoresis gel with the <i>Hin</i>fI clones digestion for mCherry, GFP, LacI TetR with pSB1C3 vector as control, Lambda-<i>Pst</i>I as marker.</span> <br>
+</figure>
+</center>
 </p>
 <br>