Team:BU Wellesley Software/Notebook/AlbertoNotebook

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2011 IGEM Wet Lab Notebook

Weekly log by Alberto Purwada

6/19/2011-6/20/2011

Based on the meeting, I plan to work mostly on part/device creation, especially on part characterization and data entry for the BU Registry. It is also important to note that we need to figure out how to put more media files into the online notebook, label the gel, and organize a clearer division of labor. Professor Densmore also mentioned that we should write basic laboratory protocols as future references for new people, verification method to determine if things are working, and troubleshooting method in case the experiment yields unusual result.

Using the previously prepared GFP (BBa_J52028) + Terminator (BBa_B0015) composite part, we did restriction digest them using the enzyme X and P. Since the prediction was that the GFP+Terminator will be cut away from the rest of the backbone, we expected the gel electrophoresis to produce a ~700 bp fragment (GFP+Terminator) and a ~3000 bp fragment (the backbone). However, the gel did not produce the expected result. While we saw a faint second band, it was too close to the first one, meaning that this band was too big to be a GFP+Terminator fragment.

6/13/2011-6/19/2011

GFP + Terminator

The starting materials were GFP (BBa_J52028) and Terminator (BBa_B0015). Each of these biobrick underwent restriction digest to cut out the necessary part. Because the GFP gene is large enough to be separated in gel electrophoresis, it was cut out from the backbone with EcoRi and SpeI. In contrast, the terminator is too small for gel separation so it was not separated from the backbone. Instead, the backbone was cut with EcoRI and XbaI. For the first time, the gel electrophoresis yielded visible results where the cut biobrick plasmid appeared as two bands (the cut region and the leftover). Nanodrop quantification showed that the DNA concentration for each sample was very small as it was between 2-3 ng/uL. The GFP gene and the backbone, which contains the terminator, were combined through ligation reaction.

Throughout these processes, three ligation reactions were made and thus three petri dishes were prepared for each of them. The backbone used here also contains genes that encode for both kanamycin and ampicillin resistance, so petri dishes with either antibiotic can be used for transformation. But since there had been problems with the ampicillin plate where it did not selectively inhibit the bacteria that were not transformed (this was known when the negative control in other experiment yielded positive result), kanamycin petri dishes were used. On the next day, only one out of the three dishes contained bacteria cultures (there were 4 cultures, to be exact). Plasmid prep was done and 12 hours later, the opaqueness from each tube showed that a good number of bacteria amplification had taken place. No string-like substance was found at the end of the pipet tip in each tube. Miniprep was done and the results were: Comps A 48.4 ng/uL, 260/280: 1.80, 260/230: 0.89 (composite part 1) and 14.7 ng/uL, 260/280: 1.76, 260/230: 0.91 (composite part 2). The plasmids were kept in the -20C freezer and the corresponding plasmid preps had glycerol added before being stored in 2 places.

Isolated Parts

From the recently arrived spring 2011 IGEM plate, the following parts were isolated: Bba_E0430 (RBS+YFP+Promoter), Bba_E0240 (RBS+GFP+Promoter), Bba_R2000 (Promoter that works best with the previous composite parts), Bba_E0020 (ECFP), Bba_E0032 (EYFP), and Bba_K156010 (BFP).

Miscellaneous

TAE buffer was prepared from the 50X TAE stock. Approximately 40 LB+Ampicillin agar plates were made because many of the parts contained gene that encode for Ampicillin resistance. Almost all used glasswares were autoclaved and kept for future use. Lab bench was cleaned with 70% ethanol and paper towel. QIAcube was received for demo and would stay for approximately one week from Tuesday (6/14).

6/6/2011-6/10/2011

BFP2

Plasmid prep were created from the BFP2 culture plate and incubated for approximately 16 hours for plasmid amplification. The resulting plasmids were isolated by miniprep and quantified with nanodrop. The DNA concentration in the two samples are 16.5 ng/uL and 14.9 ng/uL, respectively. While such values are below the normally acceptable range of 20 and above, we still run electrophoresis on them. No band was detected in the gel.

Pbad

Pbad promoter biobrick was obtained from the spring 2010 IGEM plate, and transformed into E. coli bacteria. The bacteria were then plated onto agar-based LB+Ampicillin growth medium. Lots of cultures were found on the next day after the overnight transformation. Instead of uniform appearance, however, each plate was composed of both dark or light colonies. Plasmid prep was done for each of the color type (pipet tip was used instead of metal loop) and each tube was incubated for 12 hours. On the next morning, the tip in each tube was found to contain a spiral, string-like thing and most of the plasmid preps were not opaque. After the miniprep, each of the plasmid was quantified with nanodrop. Their respective concentrations were 10.0 ng/uL and 8.3 ng/uL. They were also found to be free from contaminants (based on the 260/280 and 260/230 values). But none of the samples appeared on the gel after electrophoresis.

In order to understand the problem that had been plaguing our work, we went to a biology lab to view the plasmid preps under compound microscope and conduct gram staining analysis. All plasmid preps seemed to be contaminated with either another type of bacteria (due to differences in shape and size) or unidentified string-like compound. Many of the plasmid preps contain purple-colored bacteria, even though E. coli bacteria are supposed to be pink since they are gram-negative.

Based on these observations, we plan to improve on the lab cleanliness and avoid contamination. One important decision here was to autoclaved all of the pipet. This was done not only to avoid the possibility of contamination in daily experiments but also to ensure that the plasmid preps will properly amplify the plasmid-containing bacteria.

@@ Line 6: / Line 6: @@
   | __FORCETOC__
   |}
+<h2><br> 6/19/2011-6/20/2011 </h2>
+Based on the meeting, I plan to work mostly on part/device creation, especially on part characterization and data entry for the BU Registry. It is also important to note that we need to figure out how to put more media files into the online notebook, label the gel, and organize a clearer division of labor. Professor Densmore also mentioned that we should write basic laboratory protocols as future references for new people, verification method to determine if things are working, and troubleshooting method in case the experiment yields unusual result.
+Using the previously prepared GFP (BBa_J52028) + Terminator (BBa_B0015) composite part, we did restriction digest them using the enzyme X and P. Since the prediction was that the GFP+Terminator will be cut away from the rest of the backbone, we expected the gel electrophoresis to produce a ~700 bp fragment (GFP+Terminator) and a ~3000 bp fragment (the backbone). However, the gel did not produce the expected result. While we saw a faint second band, it was too close to the first one, meaning that this band was too big to be a GFP+Terminator fragment.
 <h2><br> 6/13/2011-6/19/2011 </h2>
@@ Line 17: / Line 23: @@
 *Isolated Parts
-From the recently arrived spring 2011 IGEM plate, (to be continued)...
+From the recently arrived spring 2011 IGEM plate, the following parts were isolated: Bba_E0430 (RBS+YFP+Promoter), Bba_E0240 (RBS+GFP+Promoter), Bba_R2000 (Promoter that works best with the previous composite parts), Bba_E0020 (ECFP), Bba_E0032 (EYFP), and Bba_K156010 (BFP).
 *Miscellaneous