Team:NRP-UEA-Norwich/Week5

From 2012.igem.org

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=Week 5=
=Week 5=
-
==Day 1==
+
Again, this week was very much centered around lab work and in producing our first BioBricks. The PCR reactions gave us a good starting point and we made great progress that was rewarded at the end of the week where we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first BioBricks!
 +
We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.
 +
 +
==Day 1 (06/08/12)==
 +
 +
===Research===
-
. Rachel and Russell ran their RFP/eCFP/AraC isolated plasmids on an agarose gel to see if they were there. The results of the gel seemed unusual.
+
With the constructs for the comparator circuit designed, research went ahead on how and who we would get the constructs synthesised. Khadija and Pascoe looked at various companies to find one which would synthesise our DNA at the best price but also within a small time frame.
-
==Day 2==
+
===Labs===
 +
[[File:Russel+Rachel_gel.png | thumb | '' '''Figure 1.''' Gel electrophoresis of plasmid isolations of various fluorescent BioBricks (RFP, eCFP and AraC promoter)'']]
-
. Rachel and Russell calculated the expected length of plasmid and found that the plasmids on the agarose gel were completely different to expected. Decided to carry out a restriction digest of the plasmids to remove the inserts and then run those on a gel to find out if they are as expected. After a nanodrop we found that there was a very small amount of DNA and likely too little to work with; mini-prepped the second samples for each biobrick and nanodropped them.
+
Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands found were not in-keeping with the size they should be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed (''Figure 1.''). Further validation experiments were discussed.
-
==Day 3==
+
A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.
-
==Day 4==
+
The PyeaR + GFP BioBrick in pSB1C3, that had been miniprepped last week, was nanodropped. Following Friday, when the restriction digest of the plasmid with EcoRI and PstI was unsuccessful, we repeated the digest to remove the insert from the backbone. Joy used the nanodropped Pyear + GFP sample that had been validated. Unlike Friday, the digest was successful!
 +
All protocols helpful for purpose recreation of results can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol here].
-
==Day 5==
+
==Day 2 (07/08/12)==
 +
 
 +
===Research===
 +
 
 +
Continued research into companies for synthesising our DNA. Khadija emailed GenScript for a quote of our order and time it would take for the genes to arrive.
 +
 
 +
===Labs===
 +
 
 +
Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter BioBricks in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the samples and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:
 +
 
 +
 
 +
RFP = 20.0 ng/μl  |  eCFP = 2.9 ng/μl  |  AraC1 = 16.0 ng/μl  |  AraC2 = 2.9 ng/μl
 +
 
 +
 
 +
Clearly a significant difference between the expected and actual result. Following this experiment, Russell carried out a DNA mini-prep on the second cell sample left in the fridge last week, as a backup, his time using the Bioline Plasmid Isolation kit (Protocol can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Isolating_DNA_.28Bioline.29 here]). The plasmids that had been isolated from the second DNA mini-prep were then nano-dropped and showed slightly better results, which were:
 +
 
 +
 
 +
RFP = 28.3 ng/μl  |  eCFP = 46.9 ng/μl  |  AraC1 = 7.4 ng/μl  |  AraC2 = 17.8 ng/μl
 +
 
 +
 
 +
Despite not being in the 100s of ng/μl as desired, we decided that we could use this DNA to carry out future restriction digests and gel electrophoresis in order to validate. We then isolate DNA from the ''E. coli'', that was to be transformed by Rachel for the future restriction digest/ligation, to produce new BioBricks.
 +
Rachel transformed fresh "E. coli" with the original BioBricks in order to produce a new stock of DNA, as we had left gaps between using the bacteria and DNA previously, which could account for the very low levels of DNA we had experienced in the nanodrops.
 +
 
 +
 
 +
==Day 3 (08/08/12)==
 +
 
 +
===Labs===
 +
[[File:Glow_2.png | thumb | ''An example of a successful transformation with the RFP BioBrick'']]
 +
 
 +
Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.
 +
 
 +
Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the BioBricks transformed into ''E. coli'' by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.
 +
 
 +
A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].
 +
 
 +
The restriction digests (see Friday, Week 4) of the repeated PCR of BM/MB was perfromed. The samples were run on a gel and the results proved that the PCR was successful in amplifying the hybrid promoters!
 +
 
 +
Following validation of the PCR products, we [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Ligation ligated] MB and BM promoters into pSB1C3. We initially nanodropped each of the samples and used the quantitate data to set up a ligation at a 3:1 ratio between insert to pSB1C3. Positive and negative controls were added to ensure quality control if something did go wrong, so that we could determine the source of the error. As a negative control, pSB1C3 would be transformed into alpha gold select cells from Bioline; as a positive control, we ligated PyeaR-GFP into pSB1C3 and transformed the plasmid into alpha cells.
 +
 
 +
==Day 4 (09/08/12)==
 +
 
 +
===Research===
 +
 
 +
We finally have our constructs ready to send of for synthesising!
 +
 
 +
===Labs===
 +
 
 +
Rachel and Russell looked at the transformed bacteria and found that the plates had been contaminated. We had some of the transformants left over, so decided to plate them again. However the plates that were made, were dry/thin. We decided to postpone the repeat of the experiment and prepare new plates the next day.
 +
 
 +
To test whether the ligation had worked, a restriction digest was run. We digested the samples with BamH1 to linearise them. If the ligation was successful, we were expecting the inserts to be in pSB1C3 and hence be 2321bp in size. If unlinearised, the sample should travel further due to supercoiling. When linearised they should match this length. Running the products on the restriction digest on an agarose gel, showed that we did indeed succeed in ligating BM and MB into the iGEM backbone!!!
 +
 
 +
The potential BioBricks were then transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.
 +
 
 +
The pilot study to measure the growth rate of Pyear cells was carried out for the planned 6 hours. From a plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. To this, potassium nitrate was added at 0mm, 5mM and 10mM concentration. ''E.coli'' cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. To generate a calibration curve, as we did not know the number of viable cells in relation to the absorbance at 660 nm, the initial cell sample was plated as well as dilutions of it. The absorbance of various dilutions of the original cell culture was measured and the data combined to generate a calibration curve.
 +
 
 +
==Day 5 (10/08/12)==
 +
 
 +
===Research===
 +
 
 +
After some discussion with advisors into finance, feasibility of time and work, we decided to only send one pair of our constructs to be synthesised.
 +
 
 +
===Labs===
 +
 
 +
No growth was observed of the cells transformed with the ligation product of BM/MB PCR in pSB1C3.  We suspected an issue with the agar as it looked again dry and thin. We still had some bacteria and DNA left from the previous transformation and were able to re-transform with new, fresh agar plates to double check of the transformation was successful.
 +
 
 +
Russell and Rachel plated their transformed bacteria and incubated them over night.
 +
 
 +
The plates from the previous day's pilot study of PyeaR + GFP were viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies. Results of the adjusted protocol can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_study_of_growth_of_PyeaR_in_different_concentrations_of_potassium_nitrate here].

Latest revision as of 00:16, 27 September 2012

Header1NewGreen.png

NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Contents

Week 5

Again, this week was very much centered around lab work and in producing our first BioBricks. The PCR reactions gave us a good starting point and we made great progress that was rewarded at the end of the week where we successfully ligated the BM and MB inserts into the plasmid backbone. We had our first BioBricks! We also began our growth study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.

Day 1 (06/08/12)

Research

With the constructs for the comparator circuit designed, research went ahead on how and who we would get the constructs synthesised. Khadija and Pascoe looked at various companies to find one which would synthesise our DNA at the best price but also within a small time frame.

Labs

Figure 1. Gel electrophoresis of plasmid isolations of various fluorescent BioBricks (RFP, eCFP and AraC promoter)

Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands found were not in-keeping with the size they should be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed (Figure 1.). Further validation experiments were discussed.

A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.

The PyeaR + GFP BioBrick in pSB1C3, that had been miniprepped last week, was nanodropped. Following Friday, when the restriction digest of the plasmid with EcoRI and PstI was unsuccessful, we repeated the digest to remove the insert from the backbone. Joy used the nanodropped Pyear + GFP sample that had been validated. Unlike Friday, the digest was successful! All protocols helpful for purpose recreation of results can be found here.

Day 2 (07/08/12)

Research

Continued research into companies for synthesising our DNA. Khadija emailed GenScript for a quote of our order and time it would take for the genes to arrive.

Labs

Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter BioBricks in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC). However the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1). A stark difference from we had expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, the other one in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the samples and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:


RFP = 20.0 ng/μl | eCFP = 2.9 ng/μl | AraC1 = 16.0 ng/μl | AraC2 = 2.9 ng/μl


Clearly a significant difference between the expected and actual result. Following this experiment, Russell carried out a DNA mini-prep on the second cell sample left in the fridge last week, as a backup, his time using the Bioline Plasmid Isolation kit (Protocol can be found here). The plasmids that had been isolated from the second DNA mini-prep were then nano-dropped and showed slightly better results, which were:


RFP = 28.3 ng/μl | eCFP = 46.9 ng/μl | AraC1 = 7.4 ng/μl | AraC2 = 17.8 ng/μl


Despite not being in the 100s of ng/μl as desired, we decided that we could use this DNA to carry out future restriction digests and gel electrophoresis in order to validate. We then isolate DNA from the E. coli, that was to be transformed by Rachel for the future restriction digest/ligation, to produce new BioBricks. Rachel transformed fresh "E. coli" with the original BioBricks in order to produce a new stock of DNA, as we had left gaps between using the bacteria and DNA previously, which could account for the very low levels of DNA we had experienced in the nanodrops.


Day 3 (08/08/12)

Labs

An example of a successful transformation with the RFP BioBrick

Rachel checked the development of the samples of transformed bacteria and found that some had grown but didn't quiet show as much growth as expected; they were left to incubate for a few more hours to grow before inoculation.

Russell carried out a restriction digest of each isolated plasmid, of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He performed single digests with just EcoRI to linerarise the plasmid and double digests with EcoRI + PstI to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA, due to the unusual results previously. If the gel showed fragments of expected sizes then the BioBricks transformed into E. coli by Rachel will be used in the ligation of reporters to the hybrid promoter and to carry out quantitative experiments.

A plan was made to characterise PyeaR + GFP (BBa_K1001) and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use. The final studies and results can be found here.

The restriction digests (see Friday, Week 4) of the repeated PCR of BM/MB was perfromed. The samples were run on a gel and the results proved that the PCR was successful in amplifying the hybrid promoters!

Following validation of the PCR products, we ligated MB and BM promoters into pSB1C3. We initially nanodropped each of the samples and used the quantitate data to set up a ligation at a 3:1 ratio between insert to pSB1C3. Positive and negative controls were added to ensure quality control if something did go wrong, so that we could determine the source of the error. As a negative control, pSB1C3 would be transformed into alpha gold select cells from Bioline; as a positive control, we ligated PyeaR-GFP into pSB1C3 and transformed the plasmid into alpha cells.

Day 4 (09/08/12)

Research

We finally have our constructs ready to send of for synthesising!

Labs

Rachel and Russell looked at the transformed bacteria and found that the plates had been contaminated. We had some of the transformants left over, so decided to plate them again. However the plates that were made, were dry/thin. We decided to postpone the repeat of the experiment and prepare new plates the next day.

To test whether the ligation had worked, a restriction digest was run. We digested the samples with BamH1 to linearise them. If the ligation was successful, we were expecting the inserts to be in pSB1C3 and hence be 2321bp in size. If unlinearised, the sample should travel further due to supercoiling. When linearised they should match this length. Running the products on the restriction digest on an agarose gel, showed that we did indeed succeed in ligating BM and MB into the iGEM backbone!!!

The potential BioBricks were then transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.

The pilot study to measure the growth rate of Pyear cells was carried out for the planned 6 hours. From a plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. To this, potassium nitrate was added at 0mm, 5mM and 10mM concentration. E.coli cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. To generate a calibration curve, as we did not know the number of viable cells in relation to the absorbance at 660 nm, the initial cell sample was plated as well as dilutions of it. The absorbance of various dilutions of the original cell culture was measured and the data combined to generate a calibration curve.

Day 5 (10/08/12)

Research

After some discussion with advisors into finance, feasibility of time and work, we decided to only send one pair of our constructs to be synthesised.

Labs

No growth was observed of the cells transformed with the ligation product of BM/MB PCR in pSB1C3. We suspected an issue with the agar as it looked again dry and thin. We still had some bacteria and DNA left from the previous transformation and were able to re-transform with new, fresh agar plates to double check of the transformation was successful.

Russell and Rachel plated their transformed bacteria and incubated them over night.

The plates from the previous day's pilot study of PyeaR + GFP were viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies. Results of the adjusted protocol can be found here.