Team:NRP-UEA-Norwich/Week4

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=Week 4=
=Week 4=
-
==Day 1==
+
This week we made great progress into designing our own constructs for the comparative circuit. Many challenges in the design of the complementary sequences were overcome such as the creation of scars and the standard we would design it for. The amount of lab work had definitely picked up. We are extremely close to creating our own first BioBricks. Hopefully by next week, we will have them.
-
===Research and Meetings===
+
==Day 1  (30/07/12)==
-
. Khadija and Pascoe found problem over the weekend with stop codon; two options are scaffold or assembly standard 23. Leaning towards the other assembly standard as it's less risky than the scaffold, however discussion occuring on both. They developed two main constructs and each took one; one saw overlapping stands, the other saw strands together. Khadija produced a logic table of codons that could bind to one another.
+
===Research and Meetings===
-
. Amy came to visit in order to look at the studio for our film being recorded on Wednesday.
+
Khadija and Pascoe encountered obstacles with stop codons over the weekend and two possible solutions were thought up. One was a scaffold construction, the other one the assembly using standard 23. The team leaned towards the assembly 23 standard as it's less risky than the scaffold, however was decided that further research is needed in order to make an informed decision.
 +
Two main constructs were developed; one saw overlapping stands, the other saw strands together. Khadija produced a logic table of codons that could bind to one another. Amy also came to visit in order to look at the studio we bfor our film being recorded on Wednesday.
===Labs===
===Labs===
-
. Joy and Lukas looked at doing the ligation for our first biobrick using pYeaR/CarG promoter.
+
Joy and Lukas continued planning the ligation of BM/MB promoter into the pSB1C3 backbone which will give us our first two BioBricks. As the B-M fragment with the correct prefix and suffix endings was ready from last week, the [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Ligation iGEM protocol] was performed.  
-
. Rebecca transformed the ligated biobricks.
+
Following the ligation, Rebecca transformed the potential BioBrick into Bioline competent alpha cells and the cell cultures were grown on the appropriate media (now containing 100ug/µl of chloramphenicol) at 37 °C over night.
 +
Observing cells transformed on Friday (that were stored in the fridge after the initial incubation for 24 hours), Russell and Rachel found that the cultures had not grown as much as they would have liked. Therefore the plates were returned to the 37 °C incubator to grow some more overnight.
-
After coming into the lab following growth overnight on the previous Friday to Saturday Russell and Rachel found that the transformed ''E. coli'' had not grown as much as they would have liked, therefore the plates were returned to the 37 °C incubator to grow some more overnight.
+
==Day 2 (31/07/12)==
-
 
+
-
==Day 2==
+
===Research===
===Research===
-
. Russell and Rachel looked at nitrite reductase sequences.
+
Russell and Rachel researched nitrite reductases (NOSs) and their sequences, to be able to determine it functionality as a reporter in the NO sensing gene construct and its ability to raise NO levels to toxic concentrations. In addition the sequences were observed to judge whether it would be feasible to get them synthesised.  
-
 
+
The whole team worked together with Khadija and Pascoe on the complementary DNA sequences for the comparative circuit; four members helping with Khadija's logic map and three with Pascoe's idea.
-
. The whole team worked with Khadija and Pascoe on the DNA sequence, four working on Khadija's logic map and three working with Pascoe's idea.
+
===Labs===
===Labs===
-
. As the plates from yesterday's transformations showed no growth, Rebecca redid two of the transformations, this time with both negative and positive controls. She also made more chlorophenicol plates and inoculated more hybrid promoter transformed ''E.coli'' into media culture. The transformations from yesterday were incubated for longer in hope that colonial growth would appear.
+
As the colonies transformed with the ligation product of B-M in pSB1C3 from yesterday showed no growth, Rebecca redid transformation using the same ligation product and incorporated untransformed alpha cells as a negative control and chloramphenicol resistant PyeaR cells as a positive controls.
 +
 +
More chloramphenicol plates were poured to stock pile for the transformation of ligation products in the coming weeks. The transformants from yesterday were incubated for longer in hope that colonial growth would appear.
-
. Lukas and Joy isolated plasmid DNA from "E.coli" containing the hybrid promoter 1. They then double digested the plasmid using EcoR1 and Pst1. Sac1 and Spe1 was also used as a test for the correct plasmid isolated. This was also carried out on hybrid promoter 2 plasmids. Following this, they ran the digested plasmid fragments of promoter 1 on gel.
+
Lukas and Joy isolated plasmid DNA from "E.coli" containing the hybrid promoter MB plasmid. They then double digested the DNA using EcoRI and PstI to separate the MB promoter with iGEM prefix and suffix attached. Double digests with SacI and SpeI of the same plasmid were also performed as validation of the DNA. Both digests were run on an agarose gel (''Figure 1.''). [[File:M-B_in_pUC57_E+P_S2.png | thumb | '' '''Figure 1.''' Gel electrophoresis of B-M in pUC57, double digested with EcoRI and PstI as well as validated with SacI and SpeI'']]
 +
The protocols can be seen [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Gel_Electrophoresis here.]
 +
The validation digest was also carried out on the BM plasmid, that had been used in the ligation on Monday, to exclude the quality of the starting material of the transformation as a source of error. 
 +
After incubating the ''E. coli'' cultures, that had been transformed with RFP, eCFP and AraC promoter, for longer there was some success as more colonies were oberved on almost all the plates. Russell and Rachel inoculated 5 ml tubes of LB media (+ 5µl ampicillin) with transformed ''E. coli'' as followed:
-
After leaving the ''E. coli'' that had been transformed with RFP, eCFP and AraC promoter to grow some more overnight there was some success as more colonies were produced on almost all of plates. Russell and Rachel inoculated 5 ml tubes of LB media (+ 5µl ampicillin) with transformed ''E. coli'' as follows:
+
Two tubes of RFP-transformed ''E. coli'' from different colonies on the 200µl plate
-
. Two tubes of RFP-transformed ''E. coli'' from different colonies on the 200µl plate
+
Two tubes of eCFP-transformed ''E. coli'' from different colonies on the 200µl plate
-
. Two tubes of eCFP-transformed ''E. coli'' from different colonies on the 200µl plate
+
Four tubes of AraC Promoter-transformed ''E. coli'' from different colonies on the 200µl plate (due to the fact that promoters were needed for each of the fluorescent proteins,  we decided to create double the amount).
-
. Four tubes of AraC Promoter-transformed ''E. coli'' from different colonies on the 200µl plate (due to the fact we need promoters for each of the fluorescent proteins thus we decided to create double the amount).
+
The inoculated media was placed in the 37 °C agitator and left overnight to grow. The protocol for this can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Inoculations Here]
-
The media inoculated media was placed in the 37 °C agitator and left overnight to grow. The protocol for this can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Inoculations Here]
+
==Day 3 (01/08/12)==
-
 
+
-
==Day 3==
+
[[File:CombinedMRNA.png | thumb | The JK-R5-PL Construct]]
[[File:CombinedMRNA.png | thumb | The JK-R5-PL Construct]]
-
. Again the transformation was unsuccessful.
+
===Labs===
-
. Rebecca purified hybrid promoter 2 from gel slice.
+
Rachel and Russell checked the inoculated media from the day before and observed that bacterial growth had not yielded enough cells to perform a likely successful experiments. Therefore it was decided to continue the incubation before attempting a DNA isolation via mini prep.
-
. Lukas ran hybrid promoter 2 on gel.
+
Repeat transformations of BM/MB in pSB1C3 did not yield any viable cells. But the negative control showed no growth and the positive controlled did, from which it was concluded that the error must lie with the ligation and not with the transformation.
-
===Labs===
+
Rebecca purified the BM insert (cut from pUC57 with EcoRI and PstI, see ''Figure 2.''), cut out from the agarose gel slice and frozen the previous night. This was performed using a Promega gel purification kit to minimise the error that might be introduced with the Bioline kit. The protocol can be found on the [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol lab protocol page]. The isolated sample was run on an agarose gel. [[File:Bm_dd,_E+P,_Sx2.png | thumb | '' '''Figure 2.''' B-M in pUC57, double digested with EcoRI and PstI and validated with SpeI and SacI'']]
-
Rachel and Russell checked the inoculated media and found that it had grown a little bit but not enough for a likely successful experiment, therefore they chose to leave the bacteria to grow some more before attempting a DNA mini-prep.
 
===Research===
===Research===
-
In the morning Khadija, Pascoe, Rebecca and Russell looked at one of the proposed constructs and worked at producing a DNA sequence that would give us two complimentary strands that would bind together once transcribed to mRNA. After a few hours of work and lots of adjustments and edits we finally had our construct which we named the JK-R5-PL construct (using the initials of the team members). The construct is the coming-together of two separate constructs which would be in between the promoter and reporter sequences of the gene we were producing. We were pleased with the JK-R5-PL construct because it meant the ribosome binding site of both original constructs was covered, meaning that the two reporter proteins would not be expressed resulting in the first step of the comparator circuit being completed!
+
In the morning Khadija, Pascoe, Rebecca and Russell looked at one of the proposed constructs and worked on producing a DNA sequence that forms two complimentary strands that would bind together once transcribed to mRNA. After a few hours of design and lots of adjustments and edits, we finally had our construct which we named the JK-R5-PL construct (using the initials of the team members). The sequence is the coming-together of two separate constructs, which would be in between the promoter and reporter sequences of the gene we were producing. We were pleased with the JK-R5-PL construct because it meant the ribosome binding site of both original constructs were covered, resulting in the two reporter proteins not to be expressed. This would ultimately result in the first step of the comparator circuit being completed!
===Video===
===Video===
-
Amy Congdon visited along with two of her colleagues to film for the final presentation video. The artist team had produced some fantastic visual adaptations of cancer cells and the bacteria climbing over them to analyse the environment and act upon the information received. We also filmed the more concept-based aspect of the video, which looks at a future world where comparator circuit-containing bacteria are commonplace in all human beings assessing the body for disease and reporting the results back to the person.
+
Amy Congdon visited along with two of her colleagues to film for the final presentation video. The artist team had produced some fantastic visual adaptations of cancer cells and the bacteria interacting with them while analysing the environment and act upon the information received. We also filmed the more concept-based aspect of the video, which looks at a future world where comparator circuit-containing bacteria are commonplace in all human beings, assessing the body for disease and reporting the results back to the person.
-
==Day 4==
 
-
. RECEIVED A LETTER FROM DAVID ATTENBOROUGH!
+
==Day 4 (02/08/12)==
-
. Posters for the Forum returned from the print, ready to start putting them up.
+
WE RECEIVED A LETTER FROM DAVID ATTENBOROUGH!!!
-
. Russell and Rachel worked on the DNA purification and isolated the plasmids for RFP, eCFP and AraC promoter and put on ice to run on an agarose gel the next day.
+
[[File:Letter.jpg | thumb | Woooooooo!!!]]
 +
 
 +
 
 +
Posters for our human outreach event at the Forum were returned from the print shop and we were getting ready to start putting them up.
 +
 
 +
===Labs===
-
. Joy, Rebecca and Lukas ran a PCR of the plasmid backbone to get some more.
+
As the gel purification of MB/BM fragments did not yield DNA samples of high concentrations and we suspected that this might be the cause of the failed ligations,  Joy  performed a PCR on the MB/BM sequences in pUC57 to increase the amount of DNA we had at hand. The PCR was performed with a negative control and it is important to note that the primers used were standard amplification primers for the pUC57 backbone which allowed us to amplify the hybrid promoters as well as the iGEM suffix and prefix.
-
==Day 5==
+
Russell and Rachel proceeded with DNA isolations of the cells cultures containing the RFP, eCFP and AraC promoter BioBricks. The samples were put on ice for validation gel electrophoresis the next day. A Promega plasmid isolation kit was used and for each original cell culture, a small amount of unused cells was stored in the fridge as a back-up in case of any problems with the plasmid isolation.
-
. Ran a gel for PCR and isolated RFP/eCFP/AraC
+
==Day 5 (03/08/12)==
-
. Nano-dropped the plasmid DNA
+
To check DNA concentrations of BM and MB PCR products, nano drops were performed. We found that the DNA concentrations off all products were above 300ng/µl. To purify the samples and prevent primers, salt and buffers from interfering with downstream applications of the MB/BM genes, a Promega PCR cleanup kit was used. [[File:PCR_double_digest.png | thumb | '' '''Figure 3.''' PCR product of B-M/M-B in pUC57, digested using BanHI'']]
-
. Restriction digest of insert + primers and plasmid backbone
+
Lukas carried out a double (EcoRI + PstI) and single (EcoRV) restriction digest on PyeaR + GFP (BBa_K381001). The single digest was used to validate whether the isolated plasmid DNA is in fact BBa_k381001. The double digest would yield a linearised pSB1C backbone and fragments of the PyeaR + GFP BioBrick. They will be utilised as a positive control in ligations and transformations. Gel electrophoresis was used to visualise the DNA fragements. The single digest was successful linearising the plasmid and thus confirming that the DNA, contained in the samples, was in fact PyeaR + GFP. The double digest was not successful and needed to be repeated.
-
. Gel electrophesis to confirm plasmid identity
+
A double (EcoRI + PstI) and single (BamHI) digest was carried out on the PCR amplification of BM/MB. The enzymes EcoRI and PstI will release the promoter sequence from the primer sequences leaving them with sticky ends of the IGEM prefix and suffix. The single digest was used for validation.
 +
Following the restriction digest, the DNA was nanodropped to check the concentrations and then run on a 1.4% agarose gel. If the PCR cloning, purification and digestion were successful, 251bp fragments should be produced. The gel showed shadowing and was not completely clear. The gel will have to be rerun.
 +
The BamHI digested products were run on a gel. It showed 200bp fragments, suggesting that the insert was produced (''Figure 3.'').

Latest revision as of 00:32, 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 4

This week we made great progress into designing our own constructs for the comparative circuit. Many challenges in the design of the complementary sequences were overcome such as the creation of scars and the standard we would design it for. The amount of lab work had definitely picked up. We are extremely close to creating our own first BioBricks. Hopefully by next week, we will have them.

Day 1 (30/07/12)

Research and Meetings

Khadija and Pascoe encountered obstacles with stop codons over the weekend and two possible solutions were thought up. One was a scaffold construction, the other one the assembly using standard 23. The team leaned towards the assembly 23 standard as it's less risky than the scaffold, however was decided that further research is needed in order to make an informed decision. Two main constructs were developed; one saw overlapping stands, the other saw strands together. Khadija produced a logic table of codons that could bind to one another. Amy also came to visit in order to look at the studio we bfor our film being recorded on Wednesday.

Labs

Joy and Lukas continued planning the ligation of BM/MB promoter into the pSB1C3 backbone which will give us our first two BioBricks. As the B-M fragment with the correct prefix and suffix endings was ready from last week, the iGEM protocol was performed.

Following the ligation, Rebecca transformed the potential BioBrick into Bioline competent alpha cells and the cell cultures were grown on the appropriate media (now containing 100ug/µl of chloramphenicol) at 37 °C over night.

Observing cells transformed on Friday (that were stored in the fridge after the initial incubation for 24 hours), Russell and Rachel found that the cultures had not grown as much as they would have liked. Therefore the plates were returned to the 37 °C incubator to grow some more overnight.

Day 2 (31/07/12)

Research

Russell and Rachel researched nitrite reductases (NOSs) and their sequences, to be able to determine it functionality as a reporter in the NO sensing gene construct and its ability to raise NO levels to toxic concentrations. In addition the sequences were observed to judge whether it would be feasible to get them synthesised. The whole team worked together with Khadija and Pascoe on the complementary DNA sequences for the comparative circuit; four members helping with Khadija's logic map and three with Pascoe's idea.

Labs

As the colonies transformed with the ligation product of B-M in pSB1C3 from yesterday showed no growth, Rebecca redid transformation using the same ligation product and incorporated untransformed alpha cells as a negative control and chloramphenicol resistant PyeaR cells as a positive controls.

More chloramphenicol plates were poured to stock pile for the transformation of ligation products in the coming weeks. The transformants from yesterday were incubated for longer in hope that colonial growth would appear.

Lukas and Joy isolated plasmid DNA from "E.coli" containing the hybrid promoter MB plasmid. They then double digested the DNA using EcoRI and PstI to separate the MB promoter with iGEM prefix and suffix attached. Double digests with SacI and SpeI of the same plasmid were also performed as validation of the DNA. Both digests were run on an agarose gel (Figure 1.).
Figure 1. Gel electrophoresis of B-M in pUC57, double digested with EcoRI and PstI as well as validated with SacI and SpeI

The protocols can be seen here.

The validation digest was also carried out on the BM plasmid, that had been used in the ligation on Monday, to exclude the quality of the starting material of the transformation as a source of error.

After incubating the E. coli cultures, that had been transformed with RFP, eCFP and AraC promoter, for longer there was some success as more colonies were oberved on almost all the plates. Russell and Rachel inoculated 5 ml tubes of LB media (+ 5µl ampicillin) with transformed E. coli as followed:

Two tubes of RFP-transformed E. coli from different colonies on the 200µl plate

Two tubes of eCFP-transformed E. coli from different colonies on the 200µl plate

Four tubes of AraC Promoter-transformed E. coli from different colonies on the 200µl plate (due to the fact that promoters were needed for each of the fluorescent proteins, we decided to create double the amount).

The inoculated media was placed in the 37 °C agitator and left overnight to grow. The protocol for this can be found Here

Day 3 (01/08/12)

The JK-R5-PL Construct

Labs

Rachel and Russell checked the inoculated media from the day before and observed that bacterial growth had not yielded enough cells to perform a likely successful experiments. Therefore it was decided to continue the incubation before attempting a DNA isolation via mini prep.

Repeat transformations of BM/MB in pSB1C3 did not yield any viable cells. But the negative control showed no growth and the positive controlled did, from which it was concluded that the error must lie with the ligation and not with the transformation.

Rebecca purified the BM insert (cut from pUC57 with EcoRI and PstI, see Figure 2.), cut out from the agarose gel slice and frozen the previous night. This was performed using a Promega gel purification kit to minimise the error that might be introduced with the Bioline kit. The protocol can be found on the lab protocol page. The isolated sample was run on an agarose gel.
Figure 2. B-M in pUC57, double digested with EcoRI and PstI and validated with SpeI and SacI


Research

In the morning Khadija, Pascoe, Rebecca and Russell looked at one of the proposed constructs and worked on producing a DNA sequence that forms two complimentary strands that would bind together once transcribed to mRNA. After a few hours of design and lots of adjustments and edits, we finally had our construct which we named the JK-R5-PL construct (using the initials of the team members). The sequence is the coming-together of two separate constructs, which would be in between the promoter and reporter sequences of the gene we were producing. We were pleased with the JK-R5-PL construct because it meant the ribosome binding site of both original constructs were covered, resulting in the two reporter proteins not to be expressed. This would ultimately result in the first step of the comparator circuit being completed!

Video

Amy Congdon visited along with two of her colleagues to film for the final presentation video. The artist team had produced some fantastic visual adaptations of cancer cells and the bacteria interacting with them while analysing the environment and act upon the information received. We also filmed the more concept-based aspect of the video, which looks at a future world where comparator circuit-containing bacteria are commonplace in all human beings, assessing the body for disease and reporting the results back to the person.


Day 4 (02/08/12)

WE RECEIVED A LETTER FROM DAVID ATTENBOROUGH!!!

Woooooooo!!!


Posters for our human outreach event at the Forum were returned from the print shop and we were getting ready to start putting them up.

Labs

As the gel purification of MB/BM fragments did not yield DNA samples of high concentrations and we suspected that this might be the cause of the failed ligations, Joy performed a PCR on the MB/BM sequences in pUC57 to increase the amount of DNA we had at hand. The PCR was performed with a negative control and it is important to note that the primers used were standard amplification primers for the pUC57 backbone which allowed us to amplify the hybrid promoters as well as the iGEM suffix and prefix.

Russell and Rachel proceeded with DNA isolations of the cells cultures containing the RFP, eCFP and AraC promoter BioBricks. The samples were put on ice for validation gel electrophoresis the next day. A Promega plasmid isolation kit was used and for each original cell culture, a small amount of unused cells was stored in the fridge as a back-up in case of any problems with the plasmid isolation.

Day 5 (03/08/12)

To check DNA concentrations of BM and MB PCR products, nano drops were performed. We found that the DNA concentrations off all products were above 300ng/µl. To purify the samples and prevent primers, salt and buffers from interfering with downstream applications of the MB/BM genes, a Promega PCR cleanup kit was used.
Figure 3. PCR product of B-M/M-B in pUC57, digested using BanHI

Lukas carried out a double (EcoRI + PstI) and single (EcoRV) restriction digest on PyeaR + GFP (BBa_K381001). The single digest was used to validate whether the isolated plasmid DNA is in fact BBa_k381001. The double digest would yield a linearised pSB1C backbone and fragments of the PyeaR + GFP BioBrick. They will be utilised as a positive control in ligations and transformations. Gel electrophoresis was used to visualise the DNA fragements. The single digest was successful linearising the plasmid and thus confirming that the DNA, contained in the samples, was in fact PyeaR + GFP. The double digest was not successful and needed to be repeated.

A double (EcoRI + PstI) and single (BamHI) digest was carried out on the PCR amplification of BM/MB. The enzymes EcoRI and PstI will release the promoter sequence from the primer sequences leaving them with sticky ends of the IGEM prefix and suffix. The single digest was used for validation. Following the restriction digest, the DNA was nanodropped to check the concentrations and then run on a 1.4% agarose gel. If the PCR cloning, purification and digestion were successful, 251bp fragments should be produced. The gel showed shadowing and was not completely clear. The gel will have to be rerun. The BamHI digested products were run on a gel. It showed 200bp fragments, suggesting that the insert was produced (Figure 3.).