Vhb+RBS, Vgb+RBS, Jen1+RBS, and K+RBS (K = constitutive promoter [http://partsregistry.org/Part:BBa_K081005 BBa_K081005]), was cut with S+P, and RFP+DTT, YFP+DTT, GFP+DTT and CFP+DTT was cut with X+P. The next step will be to ligate all promoters together with a fluorescent protein, in order to test them before putting them into the final construct.
Vhb+RBS, Vgb+RBS, Jen1+RBS, and K+RBS (K = constitutive promoter [http://partsregistry.org/Part:BBa_K081005 BBa_K081005]), was cut with S+P, and RFP+DTT, YFP+DTT, GFP+DTT and CFP+DTT was cut with X+P. The next step will be to ligate all promoters together with a fluorescent protein, in order to test them before putting them into the final construct.
+
+
A gel electrophoresis was performed on Vhb+RBS, Vgb+RBS, Jen1+RBS and K+RBS as well as all the fluorescent proteins + double terminator. The fluorescent protein genes were cut out and a gel extraction was performed,, as in the protocol. The promoters were purified using the PCR Purification Kit, as described in the protocol.
+
+
K+RBS were ligated together with RFP+DTT, YFP+DTT and GFP+DTT. Jen1+RBS and VGB+RBS were ligated together with GFP+DTT, and VHB+RBS were ligated together with RFP+DTT.
Miniprepped LuxR+DTT and LuxI+DTT by using the Wizard plus SV miniprep kit from Promega. Concentrations are given below:
Biobrick
Concentration [ng/µl]
LuxI+DTT
37.6
LuxR+DTT
37.0
Vhb+RBS, Vgb+RBS, Jen1+RBS, and K+RBS (K = constitutive promoter [http://partsregistry.org/Part:BBa_K081005 BBa_K081005]), was cut with S+P, and RFP+DTT, YFP+DTT, GFP+DTT and CFP+DTT was cut with X+P. The next step will be to ligate all promoters together with a fluorescent protein, in order to test them before putting them into the final construct.
A gel electrophoresis was performed on Vhb+RBS, Vgb+RBS, Jen1+RBS and K+RBS as well as all the fluorescent proteins + double terminator. The fluorescent protein genes were cut out and a gel extraction was performed,, as in the protocol. The promoters were purified using the PCR Purification Kit, as described in the protocol.
K+RBS were ligated together with RFP+DTT, YFP+DTT and GFP+DTT. Jen1+RBS and VGB+RBS were ligated together with GFP+DTT, and VHB+RBS were ligated together with RFP+DTT.
Saturday 14.07.12
Miniprepped LuxI+DTT and LuxR+DTT according to protocols. An unexpected white precipitate appeared in the column after eluting the DNA from the filter in the final centrifugation. This was discarded, and the OD of the supernatant was measured with NanoDrop. The resulting concentrations were much higher than expected, 293.8 ng/µl for LuxR+DTT and 264.9 ng/µl for LuxR+DTT.
Due to the unexpected results, new liquid cultures were made for the two samples and the miniprep procedure will be performed again on sunday or monday.
Friday 13.07.12
Isolated plasmid DNA from the fluorescense proteins RFP (<partinfo>Bba_E1010</partinfo>), YFP(<partinfo>BBa_E0030</partinfo>), GFP (<partinfo>BBa_K082003</partinfo>) and CFP (<partinfo>BBa_E0020</partinfo>) ligated with a double terminator DTT (<partinfo>BBa_B0015</partinfo>) in addition to the constitutive promoter (<partinfo>BBa_K081005</partinfo>). The concentrations of the isolated samples are given below:
Biobrick
Concentration [ng/µl]
RFP
40,9
YFP
55,5
GFP
41,1
CFP
47,6
Const. Promoter
19,8
Further work on the test construct in J5. Also, Clone was used to simulate cutting and ligating, yielding the sequence of plasmid <partinfo>pSB1A2</partinfo> assembled with constitutive promoter and RBS (<partinfo>BBa_K081005</partinfo>) using the standard BioBrick assembly method outlined at [http://partsregistry.org/Help:Assembly http://partsregistry.org/Help:Assembly].
LuxI+DTT and LuxR+DTT was transferred to liquid medium for overnight incubation. Miniprepping will be done tomorrow.
We had our weekly meeting with the advisors. A very quick summary can be found in the Dropbox folder.
Thursday 12.07.12
LuxI and LuxR were cut and ligated with the DTT terminator. LuxI+DTT and LuxR+DTT were thereafter transformed.
Colonies of the BioBricks RFP (<partinfo>Bba_E1010</partinfo>), YFP(<partinfo>BBa_E0030</partinfo>), GFP (<partinfo>BBa_K082003</partinfo>), CFP (<partinfo>BBa_E0020</partinfo>) and the constitutive promoter (<partinfo>BBa_K081005</partinfo>) where transferred to liquid medium with amp resistance.
Restriction digestion were performed on Jen1+RBS, VGB+RBS, VHB+RBS and LuxR with the enzymes SpeI and PstI. They are now ready to be ligated together with YFP+DTT and CFP+DTT, to be tested.
Wednesday 11.07.12
The BioBricks RFP (<partinfo>Bba_E1010</partinfo>), YFP(<partinfo>BBa_E0030</partinfo>), GFP (<partinfo>BBa_K082003</partinfo>) and CFP (<partinfo>BBa_E0020</partinfo>) where ligated together with the double terminator DTT (<partinfo>BBa_B0015</partinfo>), and transformed. They are incubated overnight.
Transformed a constitiutive promoter (<partinfo>BBa_K081005</partinfo>) which also contains RBS so that we save a cloning step in our test constructs.
An experiment was performed to assess the functioning of the Bacteriphage T4 lysis device (<partinfo>BBa_K112808</partinfo>). The culture of pBAD (<partinfo>BBa_K206000</partinfo>) + lysis transformed cells inoculated yesterday was divided into two aliquots of 25 mL each in capped 50 mL plastic tubes. After removing 0.5 mL from both cultures for measurement of OD pre-induction, one aliquot was induced with 240 uL 0.1 M arabinose for a final concentration of approximately 1 mM, and the same amount of distilled water was added to the other culture. After induction, both cultures were incubated at 37 C with shaking (200 rpm). OD 600 was measured with a PerkinElmer Lambda 35 spectrometer shortly before and at 0.5, 1, 2 and 3 h after induction. At 3 h, a precipitate had developed in both cultures, more being visible in the induced culture. At this point, clearing of the induced culture was plainly visible. The precipitate interferred with pipetting during the last sampling, and a small amount was transferred to the cuvettes when measuring OD of the induced sample. This may have affected the last measurements slightly.
The results of the OD measurements are shown below.
Measurements of OD (600 nm) in E. coli culture after induction of lysis device under pBAD promoter using 1 ~mM arabinose.
The plates inoculated with lysis induced and control culture yesterday were inspected. The lysis induced plates appeared to show less growth, but the number of colonies was very high on all plates and it was not possible to get an accurate count.
Designed the LuxR+HSL promoter testing circuit in J5. Designed and ordered primers for amplification of the desired genes from the colicin E1 operon in E. coli.
Tuesday 10.07.12
As the restriction cutting of RFP, YFP, GFP and CFP yesterday turned out to be a sucsess, the rest of the samples were run on gel, and the bands containing the fluorescent proteins were cut out and purified using the QIAquick Gel Extraction Kit. Concentrations are given below:
Biobrick
Concentration [ng/µl]
RFP
2,8
YFP
2,7
GFP
2,0
CFP
1,4
The two parallels of the terminator cut yesterday with E+X was purified using the QIAquick PCR purification kit. Concentrations are given below:
Parallel
Concentration [ng/µl]
A
2.1
B
1.5
Test cutting of the fluorescent protein parts (RFP, YFP, GFP and CFP) was performed. Results are shown below:
RFP (well 2), YFP (well 3), GFP (well 5) and CFP (well 6) investigated using gel electrophoresis. The lower bands of all samples containing the DNA sequences coding for fluorescent proteins, were cut out of the gel and purified.
We confirmed that the lysis device part (<partinfo>BBa_K112808</partinfo>) we have extracted from the distribution kit is functional, by testing the construct with the pBAD strong promoter (<partinfo>Bba_K206000</partinfo>) and lysis device assemmbled together. The device was activated by inducing the pBAD promoter with arabinose. Liquid culture of cells transformed with the construct was grown overnight, and 1 mM arabinose added to a 1 mL sample of the culture. After incubation at 37 C for 3 h, the induced sample was significantly clearer compared to a non-induced negative control sample, indicating lysis.
Results from testing of lysis device coupled to the arabinose induced promoter pBAD. Arabinose was added to the sample to the left. The sample to the right is the control sample.
The absorbance of the samples at 600 nm were measured with a Lambda 35 spectrometer, using LB medium as reference. The measured values for undiluted samples were:
Induced sample: 0,3584
Control sample: 1,6498
Note that the measured absorbance of the control sample is outside of the range of 0.1-0.9 that is considered to not require dilution. Above this range, samples should be diluted to ensure that Beer's law applies. As measured, this absorbance is therefore probably too low.
In order to make a more quantitative assesment of the extent of lysis, cells were plated out from both the induced and control samples, as follows: 2, 20 and 200 uL of each on LA + Amp plates, for a total of six plates.
0.5 mL of culture inoculated yesterday (sample A) was transferred to 50 mL fresh LB + Amp in an sterile Erlenmeyer flask and incubated at 37 C with shaking.
The constructs dld+RBS, Jen1+RBS, VGB+RBS and VHB+RBS where all cut once with XbaI and SpeI, and once with an enzyme that has two seats where they digest. Which enzyme used for which construct can be found in the table below.
Construct
Double digest enzyme
dld+RBS
Eco57I (AcuI)
jen1+RBS
ApaLI
VGB+RBS
ApaLI
VHB+RBS
ApaLI
The Figure below shows the gel electrophoresis for the test cutting. Green circles indicate bands that were expected, red circles indicate unexpected bands or expected bands that were missing. The yellow circles indicate bands that might be correct, but not conclusive.
Dld+RBS (well 1+5), Jen1+RBS (well 2+6), VGB+RBS (well 3+7) and VHB+RBS (well 4+8) were test cut using X+S (well 1-4),Eco57I (well 5) and ApaLI (well 6-8). They were then studied using gel electrophoresis.
Monday 09.07.12
Miniprepped pBAD+lysis, VHB+RBS, VGB+RBS, JEN1+RBS and DLD+RBS. Concentrations were measured using NanoDrop and were as follows:
pBAD+lysis: 23.5 ng/µL
VHB+RBS: 48.3 ng/µL
VGB+RBS: 40.0 ng/µL
JEN1+RBS: 35.3 ng/µL
DLD+RBS: 24.1 ng/µL
All the segments mentioned above except pBAD+lysis were subsequently tested by digestion with restriction enzymes followed by gel electrophoresis. All plasmids were cut using XbaI and SpeI as well as one other enzyme having exactly two restriction sites on the plasmid. BciVI was used on VGB+RBS, ApaLI on JEN1+RBS and Eco57I on the remaining two plasmids, DLD+RBS and VHB+RBS.
After cutting, gel electrophoresis was performed. After running for 45 minutes, the results were as follows:
Gel showing results from test cutting after 45 minutes. Green circles indicate bands that were expected, red circles indicate unexpected bands or expected bands that were missing. The yellow circle indicates a band that is probably not the one we expected.
Inoculated one colony of the pBAD + lysis transformants into two tubes with 10 mL LB + Amp each, using an inoculating needle.
Performed restriction digestion with EcoRI and SpeI using the standard protocol on the following DNA samples:
CFP (<partinfo>BBa_E0020</partinfo>)
YFP (<partinfo>BBa_E0030</partinfo>)
RFP (<partinfo>Bba_E1010</partinfo>)
GFP (+ LVA) (<partinfo>BBa_K082003</partinfo>)
DNA from the following samples were digested with EcoRI and XbaI:
Double transcriptional terminator (DTT) <partinfo>BBa_B0015</partinfo> [Amp] (Miniprep DNA from culture inoculated from colony on Amp plate]
Double transcriptional terminator (DTT) <partinfo>BBa_B0015</partinfo> [Kan] (Miniprep DNA from culture inoculated from colony on Kan plate)
After digestion, the results were visualized by gel electrophoresis, using 4 µL from each digestion mixture. Based on the gel visualization, all the digestions appear to have been sucessful.
Gel showing results from test cutting of DTT.
We performed restriction digest [1] on a constitutive promoter <partinfo>BBa_J23119</partinfo> an Lux I <partinfo>BBa_C0061</partinfo>. The constitutive promoter was cut with the restriction enzymes SpeI and PstI. Lux I was cut with the restriction enzymes EcoRI and XbaI.
The samples were run on gel, and the correct bands were cut out and extracted.Consentrations are given in the table below:
Parallel
Concentration [ng/µl]
BBa_J23119
2,6
BBa_C0061
0,9
Saturday 07.07.12
The ligated constructs of Jen1 (<partinfo>BBa_K284002</partinfo>) + RBS (<partinfo>BBa_B0034</partinfo>), Dld (<partinfo>BBa_K284003</partinfo>) + RBS (<partinfo>BBa_B0034</partinfo>), VHb (<partinfo>BBa_K258005</partinfo>) + RBS (<partinfo>BBa_B0034</partinfo>) VGB (<partinfo>BBa_K561001</partinfo>)+ RBS (<partinfo>BBa_B0034</partinfo>)and pBAD (<partinfo>BBa_K206000</partinfo>) + lysis <partinfo>BBa_K112808</partinfo> were transferred from plates to liquid media and placed in cabinet for incubation.
The the religation plate with only the backbone plasmid did also have som growth which is not what we want. We still decided to continue on with the promoter constructs. The religation of the pBad plasmid did not show any growth.
Friday 06.07.12
Transformed pBAD-lysis and pBAD religated.
A gel electrophoresis was run on the BioBricks digested yesterday (Jen1 <partinfo>BBa_K284002</partinfo>, Dld <partinfo>BBa_K284003</partinfo> and VHb <partinfo>BBa_K258005</partinfo>). The results were the same as the first gel electrophoresis, so no gel extraction was performed. The purified DNA from Thursday 05.07.12 was used further on.
Jen1, Dld, Vgb, and Vhb cut with EcoRI and SpeI were ligated together with RBS cut with EcoRI and XbaI, and the ligated plasmids were transformed to competent Dh5α cells.
Thursday 05.07.12
The gel electrophoresis of Jen1 (<partinfo>BBa_K284002</partinfo>), Dld (<partinfo>BBa_K284003</partinfo>) and VHb (<partinfo>BBa_K258005</partinfo>) was analyzed. Only two bands per column were expected, insert and backbone, but there was at least one extra at each column. The length of the genes Jen1, Dld and VHb are 998 bp, 463 bp and 137 bp, respectively, so the bands closest to the given sizes were cut out of the gel and purified using a QIAquick Gel Extraction kit. The concentrations of the purified genes are listed below:
Dld promoter: 2,7 ng/µL
Jen1 promoter: 1,6 ng/µL
VHb promoter: 4,1 ng/µL
Since there were so many bands per column in the gel electrophoresis for Jen1 (<partinfo>BBa_K284002</partinfo>), Dld (<partinfo>BBa_K284003</partinfo>) and VHb (<partinfo>BBa_K258005</partinfo>), the procedure will be done one more time. They were cut, as described in the protocols, but with half the concentration of DNA.
Gel electrophoresis was also performed on the lysis device and pBAD promoter. The expected sizes of the DNA sequences were 2079 bp for the lysis plasmid, 1785 bp for the lysis device and about 2000 bp for the pBAD promoter with backbone. This corresponds well with the observed bands. Pictures of the gels obtained from the two electrophoresis runs are included below. On both gels, the far left and far right wells contain GeneRuler 1kb DNA ladder.
Results from gel electrophoresis with DLD, JEN1 and VHB.
Results from gel electrophoresis with lysis device and pBAD promoter.
The DNA segment containing the VGB promoter was extracted from another gel according to protocols. The resulting DNA concentration was measured twice, once to 17.4 ng/µL and once to 19.6 ng/µL.
Wednesday 04.07.12
The BioBricks colicin (<partinfo>BBa_K150009</partinfo>), dld-promoter (<partinfo>BBa_K284003</partinfo>), jen1 (<partinfo>BBa_K284002</partinfo>) and VHb-promoter (<partinfo>BBa_K258005</partinfo>) were miniprepped as described in the protocol. The concentration results are listed below:
Colicin: 58,6 ng/µL
Dld-promoter: 63,8 ng/µL
Jen1-promoter: 102,1 ng/µL
VHb (oxygen-promoter): 44,9 ng/µL
The luxI BioBrick <partinfo>BBa_C0061</partinfo> was transformed. Continued work on circuit assembly using J5.
Restriction digestion was performed on dld-promoter (<partinfo>BBa_K284003</partinfo>), jen1-promoter (<partinfo>BBa_K284002</partinfo>) and VHb-promoter (<partinfo>BBa_K258005</partinfo>) as described in the protocol. All of them cut with E and S.
Restriction digest were also performed for the vgb-promoter (E + S) and RBS (E + X), and gel electrophoresis was performed to verify that the correct DNA fragment were made during digestion. The RBS digestion sample was purified using a QIAquick PCR Purification kit. Using this kit will cause small DNA fragments, like the prefix fragment between the EcoRI and XbaI sites, to be lost. All of the vgb sample was run on gel, to separate the insert to be further used, from the backbone.
Performed restriction digest on the pBAD (<partinfo>BBa_K206000</partinfo>) and lysis <partinfo>BBa_K112808</partinfo> parts, cutting pBAD with SpeI and PstI, and the lysis part with XbaI and PstI.
Tuesday 03.07.12
Wiki design updated. [http://j5.jbei.org/ J5] was used to determine how to assemble the genetic circuit. The total size of the complete plasmid containg all the parts of our system will probably be 8000-9000 bp ((depending on the size of the backbone), of which the BioBricks make up about 6500 bp.
Also, it was discovered that the colicin BioBrick (<partinfo>BBa_K150009</partinfo>) consists of a number of DNA segments, not only genes for the necessary colE1 proteins. Among these are the luxR gene and the LuxR-HSL promoter as well as a lysis-inducing component. This means that inserting the entire BioBrick into our circuit will be problematic, as the LuxR production and LuxR-HSL sensitivity of the construct will interfere with the oxygen promoter and the rest of the luxR components in our circuit. The problem can probably be circumvented if we amplify the genes coding for the two colE1 proteins and connect them to a constitutive promoter.
Dld (<partinfo>BBa_K284003</partinfo>), Jen1 (<partinfo>BBa_K284002</partinfo>) and VHB (<partinfo>BBa_K258005</partinfo>) were transferred to liquid medium.
Monday 02.07.12
Restriction digestion was performed on isolated DNA from the VGB and RBS biobricks (<partinfo>BBa_k561001</partinfo> and <partinfo>BBa_B0034</partinfo>) according to protocols. Gel electrophoresis was used to determine the size of the resulting fragments. The RBS restriction digest was filtered using the QIAquick PCR purificat kit. The results for VGB were not as expected, and restriction digest of this part was therefore performed again, this time with addition of [http://en.wikipedia.org/wiki/Calf-intestinal_alkaline_phosphatase CIP] to the digest solution at the end of the procedure.
Dld (<partinfo>BBa_K284003</partinfo>), Jen1 (<partinfo>BBa_K284002</partinfo>) and VHB (<partinfo>BBa_K258005</partinfo>) were transformed as described in protocols.
Sunday 01.07.12
Miniprepped all three tubes with pBAD inoculated yesterday. Tube 3 contained 10 mL culture and was divided into two tubes, so that all processed tubes contained ~5 mL culture. Results of DNA measurements were as follows (ng/uL):
pBAD B1: 24,0
pBAD B2: 18,9
pBAD B3-1: 11,4
pBAD B3-2: 8,1
Discarded B3-1 and B3-2, placed B1 and B2 in -20 C freezer.
In order to make a new plate of Lysis 1, transferred 20 uL from one of the tubes with liquid culture inoculated yesterday to one LA + Amp plate, and 50 uL to another. Placed the plates in the 37 C incubator cabinet. Also transferred 50 uL as inoculate to the tube used as negative control yesterday and placed it in the shaking incubator. Then combined all three tubes into one and placed in refrigerator to limit cell death before miniprep tomorrow.
Saturday 30.06.12
Removed pBAD plates from 37 C incubator. Good growth on all transformed plates, Negative control showed no growth. Inoculated three colonies from plate B in liquid LB + Amp. Discarded the "mixed" plate. Placed plates A and B in refrigerator. Also inoculated three colonies from the lysis biobrick <partinfo>BBa_K112808</partinfo> using the plate from 22/6. Used an inoculating needle for all inoculations today.
Friday 29.06.12
LuxI (<partinfo>BBa_K092400</partinfo>) was transferred to petri dishes with Chloramphenicol and Kanamycin, to test if LuxI has a different resistance than given. A new batch of LA-medium with Ampicilin was prepared and poured on to petri dishes.
Researched the ArcA protein and its binding sequence(s) to investigate whether the lldPRD operon promoter can be modified to eliminate repression by ArcA-P in the anaerobic state. Located a putative ArcA-P binding site in the promoter sequence: The sequence GTTAACTAAATGTTA is the reverse complement of the minus strand of the 85 bp promoter sequence from position 38 to 52. This sequence is identified by [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1999.01347.x/full#t1 McGuire et al (1999)] (see [http://arep.med.harvard.edu/ecoli_matrices/dat/arcA.dat this] list). In the GenBank entry this part of the sequence reads TAACATTTAGTTAAC
Favorov et al. have made a newer suggestion for a general motif for ArcA binding sites - see [http://bioinformatics.oxfordjournals.org/content/21/10/2240.full#sec-5 this article] and the computational result [http://favorov.bioinfolab.net/SeSiMCMC/examples/arcA/result.html here]. According to Favorov, the crucial features of the site is a direct repeat, as shown below:
atacaTAACatttagtTAACcattc
Extracted the pBAD strong promoter biobrick <partinfo>BBa_K206000</partinfo> and transformed it in two samples. Plated out on three Amp plates as follows: 200 uL from sample A, 200 uL from sample B, 20 uL each from sample A and B.
Thursday 28.06.12
A colony of LuxR (<partinfo>BBa_R0062</partinfo>) was transferred to liquid medium, but there were no colonies of LuxI (<partinfo>BBa_K092400</partinfo>). A new agar plate with LuxI was prepped.
Wednesday 27.06.12
Performed isolation of plasmid DNA from RBS (<partinfo>BBa_B0034</partinfo>), C78, C79 and VGF promoter (<partinfo>BBa_K561001</partinfo>). The parts coding for the LuxR and LuxI genes, <partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K092400</partinfo> respectively, were transformed again.
Tuesday 26.06.12
Colonies from <partinfo>BBa_C0078</partinfo>, <partinfo>BBa_C0079</partinfo> and <partinfo>BBa_K561001</partinfo> were transferred from agar plates to liquid medium.
Performed isolation of plasmid DNA from the ribosome binding site (RBS) part <partinfo>BBa_B0034</partinfo>, and the double transcriptional terminator (DTT) part <partinfo>BBa_B0015</partinfo> grown on ampicillin and on kanamycin. The yield was quite poor, especially for RBS. We then decided to run the spin columns one more time to see if we could get a higher yield the second time. Since we are going to use a lot of RBS, we made a liquid culture right away, so we can perform a new DNA isolation on RBS tomorrow.
Wiki design and contents was revised. Front page layout done and general layout underway.
Monday 25.06.12
Research was done on hybrid promoters in the kit and two interesting ones were found. The genes coding for the necessary activators and repressors were also included in the distribution. The resulting three biobricks of interest <partinfo>BBa_K092400</partinfo> (LuxI), <partinfo>BBa_C0078</partinfo> (C78) and <partinfo>BBa_C0079</partinfo> (C79), were transformed using the standard protocol.
The BioBrick part <partinfo>BBa_K561001</partinfo> (vgb promoter, microaerobic) was extracted from the kit, transformed into E. coli, and the transformed cells plated out on Chloramphenicol (Cm) plates.
A transformed colony containing the RBS part extracted yesterday, was transferred to liquid medium (5 mL LB)
Two transformed colonies containing the DTT part on a plasmid backbone with Ampicillin and Kanamycin reistance, which was extracted yesterday, was transferred to liquid medium as follows:
One colony grown on agar plate with ampicillin, was transferred to a tube with LB (5 mL) + Kanamycin (100 ug/mL
One colony grown on agar plate with kanamycin, was transferred to a tube with LB (5 mL) + Ampicillin (100 ug/mL)
DNA was isolated from the liquid culture of LuxR+HSL transformed cells inoculated yesterday by miniprep. The DNA concentration in the product was measured as 13,3 ng/uL.
Sunday 24.06.12
The biobricks <partinfo>BBa_B0034</partinfo> (RBS) and <partinfo>BBa_B0015</partinfo> (Double terminator, DTT) were extracted from the distribution kit and transformed into E. coli which were plated out. The liquid culture left after the plating was stored at 5 C.
Plasmid DNA was isolated from liquid cultures, inoculated on 22.06 and 23.06 respectively, of E. coli transformed with <partinfo>BBa_R0062</partinfo> (LuxR & HSL promoter) and <partinfo>BBa_K112808</partinfo> (T4 lysis device, no promoter), using Promega miniprep. The concentration of isolated <partinfo>BBa_R0062</partinfo> DNA was measured to 15,7 ng/uL. Due to the low yield, this brick may have to be regrown. Suspecting too long incubation time as the cause. The concentration of isolated <partinfo>BBa_K112808</partinfo> DNA was measured to 111,0 ng/uL. Both samples were stored in the freezer at -20 C.
To make a new liquid culture of <partinfo>BBa_R0062</partinfo> transformed E. coli, a colony from the plate made on 21.06 (stored in refrigerator at 5 C since then) was inoculated in aproximately 4.5 mL LB medium with 4.5 uL of Amp stock solution (100 mg/mL) added, and incubated with shaking at 37 C.
Saturday 23.06.12
Of two LA + Amp plates with <partinfo>BBaK112808</partinfo> transformants (20 and 200 uL) incubated since yesterday, only the 200 uL plate showed growth (10+ colonies). Negative control plate (untransformed cells) showed no growth. One colony was transferred to liquid culture.
On the 20 uL plate, a water bubble below the agar was mistaken for a colony, and a liquid culture was inoculated with a toothpick after scratching the plate. The mistake was realized, but the tube was still left to incubate, as it was thought it could act as a (weak) negative control.
Friday 22.06.12
The [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] transformant from yesterday yielded several colonies on its plate, and one colony was inoculated into liquid medium and incubated at 37 C with shaking. After adding antibiotic to the liquid medium in the growth tube, some of the medium was spilled, so the growth volume was about 2 mL.
Isolation of plasmid DNA from all 7 initial transformants (see 20.06.12) was performed with the Promega Wizard Plus SV Minipreps DNA Purification System A1460.
The biobrick <partinfo>BBa_K112808</partinfo> (Enterobacteria phage T4 Lysis Device - no promoter, called Lysis1) was extracted from the distribution kit and transformed into E. coli.
Thursday 21.06.12
We started the day with a lecture on genetic circuit modelling by Ph.d student and iGEM team instructor Marius Eidsaa, followed by a discussion of the options available in designing our system.
All 7 transformants from yesterday yielded 1 or more colonies. Negative controls (untransformed cells) plated out on Kanamycin and Ampicillin plates showed no growth, indicating that the antibiotics were effective in selecting transformants for growth. 1 colony from each of the transformants was inoculated in liquid LB medium and incubated at 37 C with shaking.
The biobrick [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] (Promoter (luxR & HSL regulated -- lux pR)) was extracted from the distribution kit and transformed using our modified version of the official iGEM transformation protocol (see Protocols).
Wednesday 20.06.12
We started the day with an introduction to computer modeling of biological sytems, and use of the Cain chemical kinetics simulation program. We then researched and discussed various biobricks, and transformed several biobricks from the iGEM DNA distribution plates into E coli.
The following biobricks (with nicknames) were extracted from the iGEM 2012 DNA distribution kit and transformed according to protocol:
Red Fluorescent Protein (RFP): [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]
Yellow Fluorescent Protein (YFP): [http://partsregistry.org/Part:BBa_E0030 BBa_E0030]
Cyan Fluorescent Protein (CFP): [http://partsregistry.org/Part:BBa_E0020 BBa_E0020]
Green Fluorescent Protein (GFP): [http://partsregistry.org/Part:BBa_K082003 BBa_K082003]
Tuesday 19.06.12
We started the day preparing lab equipment. We sterilized pipette tips, toothpicks, water and LA and LB medium. We also prepared stock solutions of ampicillin and kanamycin, and made petri dishes containing LA + Amp (100 ug/mL) and LA + Kan (100 ug/mL). Equipment and solutions were autoclaved at 120 C for 20 minutes.
We prepared LB medium (1 L) and LA medium (2 L). After mixing, each batch was divided into two bottles for autoclaving.
Stock solutions of Ampicillin and Kanamycin (both 100 mg/mL) were prepared from dry powders. After preparation and between use, the solutions were stored at -20.
After autoclaving, LA media bottles were left to cool. After reaching a temperature where they could be comfortably handled, the desired antibiotic was added using sterile technique. (500 uL stock solution to each bottle containing aproximiately 0.5 L, to a final antibiotic concentration of 100 ug/mL). The medium was then stirred and poured into petri dishes. The petri dished were left to cool down and placed cool for storage.
Monday 18.06.12
The team visited the lab and was given a EHS run-through by Merethe Christensen. We also risk evaluated the project and handed in the risk assessment, so now we are ready to start working in the lab:-)
Wednesday 13.06.12
Today, we tried to come up with a preliminary genetic circuit. We decided that using HGF as a signal molecule could be difficult, since we don't even know if a protein this size could penetrate the outer membrane and the peptidoglycan layer of E.coli. But we have found out that cancer cells excrete more lactate than healthy cells, so we decided to go for lactate, which is a small molecule. If the construct works, it could be modified to respond to other signal molecules.
Preliminary genetic construct:
Preliminary sketch of the genetic circuit. A constitutive promoter will express toxins, while promoters regulated by O2 and lactate will regulate the expression for proteins A1 and A2, which both needs to be present to start expression of the lysis genes
Friday 01.06.12
We talked about what we had found out since last week. Rolf and Jarle had since last week investigated different ways of making a cell lyse, and they found out that genes for lysis are already in this years iGEM kit.
Ove and Nina had been investigating toxins, but didn't find as much as they hoped for, but they have however found some toxins. Colisin E1 should be possible to use. They have also sent an email to Pål Fallnes, who Marit Otterlei suggested we could try to get in touch with. Apparently, he has been working quite a lot with expression of toxins in bacteria.
Gunvor and Eirin have since last week been trying to find a suitable signal molecule we could detect. Marit Otterlei suggested that we could use HGF, so Eirin and Gunvor did some reasearch around this growth factor. They found out that HGF is the only known lignad to the receptor c-Met, which is a tyrosin kinase. The idea so far is to use this, find out what signaling pathways this receptor is connected to, and find the endpoint of the signaling pathway. We assume that the endpoint is a growth factor possibly regulating a promoter, and if we find such a promoter, this could be set to control the lysis genes. The problem is that we don't know how signals are transducted to bacteria from the exterior, and this proved to be hard to find any information about. But we know that proteins resembling tyrosin kinases called BY kinases exists in bacteria, and also that proteins from eucaryotic cells have sometimes been working in bacteria when introns are removed by using mRNA and reverse transcriptase.
Both in the case of toxins we can express, and a signal molecule we could get our cells to respond to; we need more time. So we decided that Ove and Nina will continue looking for toxins for another week, and Eirin and Gunvor will investigate signal molecules more in depth.
Rahmi have found some possibly useful biobricks, and will be sending references to the biobricks in question to the team by email. Rahmi had also baked a cake for today's meeting:-)
In the case of sponsors, many of the companies we have asked request a budget. Gunvor will try to get hold of this from Eivind, and she will also talk to Merethe Christensen, who is an engineer at dept. of biotechnology, to arrange a safety excursion in the lab, which is necessary before we're allowed to work there.
We also elected Nina as our photo chief:-)
Friday 25.05.12
We started continuing our dicussion on which toxins we should make our cells produce. If we manage to make the cells lyse only in the presence of the signaling molecule we chose to go with, it shouldn't mather what the cells produce, but if the system turns out to be leaky, it will be a problem if the toxins we prodce is too toxic. That way we would also kill healthy cells. Also, another problem with too toxic molecules is that we will need a special lab to work with them. So we decided to go for the happy mean.
We also discussed the posiibility of making the intracellular concentration of toxins a checkpoint for lysis, so that even if a signal molecule is present, or the cells is experiencing an oxygen deficient environment, they would still not lyse until the concentration of anti cancer molecules is high enough.
We now have three different modules involved in our project; a production part, a detection part and a lysis part. We decided to split in three groups and investigate these modules in depth;
Production - Nina and Ove
Nina and Ove will investigate further what kind of molecules we could make our cells produce, and since we got the names of some people working with toxins on the meeting with prof. Otterlei, we decided to contact them to hear if they can help us. We will also be contacting Jay Bradner.
Detection - Eirin and Gunvor
Eirin and Gunvor will look at possible signaling molecules we could use for detection of cancer cells. So far, we have decided to investigate HGF, the growth factor prof. Otterlei suggested, and VEGF.
Lysis - Jarle and Rolf
Jarle and Rolf will look at different ways to make a cell lyse, and they will also investigate the suicide switch used by the Tokyo team.
We also decided that we need a PR chief. Rolf volunteered!
Wednesday 23.05.12
We had our first meeting with our new advisor, Marit Otterlei, and we discussed which signal molecule we could use to detect the cancer cells in addition to using the O2 promoter to detect oxygen deficient areas.
Prof. Otterlei suggested that we could use the Hepatocyte Growth Factor (abbreviated HGF), which is a growth factor regulating cell growth, cell motility and morphogenesis, that has the ability to bind to a tyrosine kinase.
Prof. Otterlei also told us that many toxins can be produced by cells, so we decided to look more at making the cells produce toxins in addition to the ones we've already talked about.
Friday 18.05.12
We began this weeks meeting with going through the to-do-list from last meeting.
Eirin:
Has concacted Christopher Anderson by email to ask how his system works, but apparently he was unnwilling to give away too many details, as his work is not yet published.
But we also came to the decision that specially because his work is still not published, we can't really be sure what his system looks like, and it is not our fault if it turns out that our systems actually work the same way, therefore, we shouldn't care too much that he is working on a similar project. But still; Rahmi and Eirin will try to get some more details out of him.
Rolf and Jarle:
Have been looking at possible sponsors. So far; we know that we will get a discount at VWR, so if we need something they sell, we should order it from them. Also, we have gotten a semi-positive response from Sigma-Aldrich.
Before next meeting, Rolf will talk to more companies.
Gunvor:
Has since the last iGEM-meeting had a meeting with Gaute Brede, who is a postdoc working in the cell biology group at institute of cancer research and molecular medicine. I tried to ask him about signal molecules we could use, and his idea was to use ATP or adenosine, as cancer cells secrete more ATP than a normal cell. This is a signal molecule that attracts the attention of the immune system, so to avoid an imune reaction, the cancer cells also secrete enzymes called ectonucleotidases, which job is to degrade ATP to adenosine, which does not attract the immune system. Also healthy cells secrete ATP, but in smaller amounts than cancer cells. And healthy cells does not secrete CD39 and CD73, which is the ectonucleotidases responsible for degradation of ATP. So in summary; since cancer cells has these ectonucleotidases, they have lower levels of ATP in their surroundings compared to healthy cells, and higher levels of adenosine in their surroundings compared to healthy cells. Also, it seems like most types of cancer cells secrete CD39 and CD873.
We discussed the possibility of using adenosine as a signal molecule, but Rhami was sceptical, as he thinks adenosine and ATP are taken up by the cells through passive transport. So that means we might have to look for another signal molecule. Since we have few ideas ourself, we decided to have a meeting early next week instead, and try to get our new advisor Marit Ottelei to attend the meeting, since she might have some ideas.
In the time leading up to the next meeting, Rolf will continue looking for sponsors, and the ones who has time should start looking at different ways to lyse a cell.
Friday 11.05.12
We had our weekly meeting. We started by going through what we have done since last week:
Eirin:
Since last week, Eirin have been reading Christopher Andersons article. It seems like several parts of his project is similar to what we are planing now. But we still don't know if the cells Anderson used lysed by a stimulus given by the cancer cells. We have to find out if he did this or not, so before the next meeting, Eirin vil send an email to ask him.
Ove:
Ove will be looking more at making a calender function, so we can see a calendar on top of the page and be able to click different dates. Gunvor suggested that he could take a look at the code the NTNU team used to generate a calendar last year.
Nina:
Nina has found several new potential anti cancer drugs that can be syntheszed by cells, but she will be looking for more potential drugs in the time leading up to next meeting.
Gunvor:
Has asked around at Institute of Physics for cancer cells we could use in some of the experiments we are planning. She talked to the engineer responsible for the cell lab, and she said that they would like to provide us with cancer cells, but it would be an advantage if we could get some bottles of cancer cells from someone who is already using them. The problem is, she is going to leave for summer vacation in the middle of june, and if she's not here, we have to start our own cell line, which means that one of us will have to spend some hours a day in the cell lab attending to the cancer cells for about two weeks, which is the time it takes from the cells have been thawed to they are ready to be used in experiments.
Since this solution doesn't seem optimal, Gunvor instead talked to a postdoc at her research group who has collaborations to researchers in the hospital. She has planned a meeting with one of her collaborators next wednesday, and Gunvor will follow her there to talk to them.
She have also sent Marit Otterlei (professor at Institute of cancer research and molecular medicine) an email, and contacted Terje Espevik, who works with confocal microscopy at the hospital, about how we can characterize the O2-promoter using fluorescence microscopy.
We also decided that we should start looking for sponsors. We will submit an application for funds to Programme of Bioinformatics (PBI), and Rolf and Jarle is going to contact VWR, Sigma-Aldrich, and Fisher Scientific.
Eivind reminded us that it is important to come up with an idea for what our genetic construct will actually look like as soon as possible, so we can start the modelling.
We decided that we will decide on an idea for a genetic circuit on next meeting, which will be on friday 18.05, at 13:30.
Have a nice weekend:-)
Wednesday 09.05.12
I have been playing around with a wiki design scheme today which can be found here. I hope we can discuss the wiki design a little bit this friday. Also I have been trying to make a calendar solution, but I haven't found any easier or more user-friendly way to implent this than to simply use the default wiki setup. So, at least for now, I think we should just keep using this site the way it is and add updates the way Gunvor did below (and I am doing now) ;) When you have added a new post to a day, you can click the button "Your signature with timestamp" in the editing menu to add your username along with the current time and date.
Thursday 03.05.12
We had a meeting, and we discussed several things we would like to look more into before we start planning what our genetic circuit will actually look like.
Here is a list of what we decided to do, and who will do it:
Check Christopher Anderson's article to see how similar his project was to ours - Eirin
Make a calendar on our wiki page - Ove
Look deeper into what kind of anti cancer drugs we could make our cells synthetize - Nina
Check if we have access to cancer cells, and find out more about molecules that are secreted form cancer cells in larger amounts than by normal cells - Gunvor
Find out more about the O2-sensitive promoter - Rahmi
Rolf was also going to do something, but I have forgotten what he was going to do...
- We also decided to have our next meeting at 13:30 next friday, and we decided to eat lunch together on wednesdays
- Rahmi came with a suggestion for characterization of the O2-sensitive promoter:
We could make the promoter control the expression of a fluorescent protein, then transform the system to cells, grow the cells on a soft medium they could migrate into, let them grow for a while, and then investigate the medium for coloured cells.
Suggestion from Gunvor: We could use TIRF (Total Internal Reflection Fluorescence) microscope, which allows us to investigate the fluorescence in a certain layer of the medium.
Wednesday 02.05.12
Gunvor and Rahmi held an introductory lecture to cloning techniques. Gunvor held a crash course in molecular biology, the biobrick concept, and the most common molecular biology techniques, while Rahmi covered more advanced cloning techniques like SLIC and Gibson. If anyone wants more information on for example SLIC and Gibson, google j5 assembly;-)
Thursday 26.04.12
Today, all the groups from last time started with giving an overview of possible project within their topic. Then we discussed for quite a long time, and in the end we decided we wanted to work with cancer. But we also decided to keep the biosynthesis of fatty acids as a side project. The electricity project turned out to be quite hard to complete in only two months, so we decided to drop it.
Our final project idea is then to make bacteria, for example E.coli cells, produce anti cancer drugs; preferably as many different molecules as possible. We have read about both enzymes and endpoints of metabolic pathways that are disadvantageous for cancer cells.
Our engineered cells should also be able to respond to a signal molecule secreted in larger amounts from cancer cells than from normal cells, for example a signaling molecule that promotes angiogenesis.
When the cells detect the signal molecule from the cancer cells, they should lyse, releasing the anti cancer molecule close to the tumour.
Another approach to reach the tumour could be to take advantage of the fact that the environment inside tumours normally is oxygen deficient. And as E.coli cells naturally migrate towards areas low in oxygen, a possible solution is also to activate the lysis gene when the cells are in an are with little oxygen.
Rahmi told us about a promoter that gets activated by low concentration of O2. This could regulate the lysis genes.
For the main project, we have to make the cells lyse at a certain concentration of an outer stimuli. For our side project it could be interresting to do the oposite; making the cells lyse when they have produced a certain amount of fatty acids.
Friday 20.04.12
Today, we decided on the overall top three projects for the team. We added the points all the team members have given to the different projects, and the list then becomes as follows:
Project
Number of points
Cancer (search and destroy)
14 points
Biosynthesis of fatty acids
10 points
Bacteria as electrical switch
6 points
Water purification - hormones
3 points
Water purification - sucralose
1 point
Oil spill removing bacteria
1 point
Biomining
1 point
We decided to look deeper into the overall top three projects. We formed three groups of two and two, and decided to look at the following things for the next meeting:
Look at earlier projects on the topic
Come up with some specific project ideas
Look at the feasibility of the proposed projects
Look at which biobricks we would need for each of the proposed projects, and see if they are present in the registry, or if we would have to make them ourselves.
The groups:
Eirin and Jarle - Biosynthesis of fatty acids
Rolf and Ove - Bacteria as electrical switch
Nina and Gunvor - Cancer (search and destroy)
We also elected Ove as wiki chief:-)
Friday 13.04.12
Since we have quite a few project ideas to choose between now, we decided that for next meeting, all team members should pick their three favourite projects. We decided to give our favourite project three points, the project on second place two points, and our third favourite project one point. Before the next meeting we will add all the points together, and se which projects we should look at more in depth.
At this meeting, we also discussed activities for Researchers Night. So far we have thought about making a construction kit to make the attendants on RN understand how we can combine different biobricks.
We also discussed bringing pipettes, letting the attendants make alginate beads with different colours that they could bring home, and bringing a microscope and coloured cells (harmless, of course).
Friday 30.03.12
We kept discussing possible projects, and we now have several projects that would be interresting to work with. Here are the topics we have discussed so far:
Fatty acid synthesis in E.coli (EDA and DHA production)
Using cells in water purification
Using bacteria to remove hormones from wastewater
Using bacteria to remove sucralose from wastewater
Be able to control pili production in order to control electron transfer between cells
Biosensor detecting cancer cells, starting synthesis of chemoterapeutic drugs when cancer cells are detected
Biosensor detecting harmful bacteria
Making bacteria destroy oil spill
Digitalize a signal from an environmental stimulus
Biomining (using microorganisms to extract metals
Friday 16.03.12
We had a meeting, where we started discussing ideas for this year's iGEM project. We also decided to eat lunch together once a week, to get to know one another.
For the outreach part of the project, Gunvor suggested that we could collaborate with Studentersamfundet to make a "lørdagsmøte" about synthetic biology.
Monday 27.02.12
We had our first meeting, and the team members met each other for the first time!
Friday 17.02.12
We recieved emails from Eivind letting us know that we are the ones that have been selected to represent NTNU in iGEM 2012. Everybody is happy!
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