Team:NTNU Trondheim/Notebook

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<h1>Notebook <small>Unfiltered notes from the lab</small></h1>
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__NOTOC__
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=Notebook=
 
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{{:Team:NTNU_Trondheim/Templates/Calendar}}
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Click on a month name or date to view the respective notebook entries. For information on plasmids and DNA mentioned in the notebook, see [https://2012.igem.org/Team:NTNU_Trondheim/Stocks Stocks] and [https://2012.igem.org/Team:NTNU_Trondheim/Constructs Constructs]. For explanations of abbreviations used, click [https://2012.igem.org/Team:NTNU_Trondheim/Abbreviations here].
 +
 +
<!--
 +
===Thursday 09.08.12===
 +
----
 +
 +
Did a restriction digest as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol] on the following parts. On the pieces to be used as inserts, the number of base pairs are also listed.
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Biobrick/Construct
 +
!BioBrick
 +
!Enzymes
 +
!Buffer
 +
!bp insert
 +
|-
 +
|LuxI+term
 +
|<partinfo>Bba_C0061</partinfo>, <partinfo>BBa_B0015</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|747
 +
|-
 +
|YFP+term
 +
|<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|852
 +
|-
 +
|Lysis
 +
|<partinfo>BBa_K112808</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|1785
 +
|-
 +
|LuxI
 +
|<partinfo>Bba_C0061</partinfo>
 +
|XbaI+PstI
 +
|3
 +
|618
 +
|-
 +
|RBS*
 +
|<partinfo>BBa_B0030</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|P<sub>luxR+HSL</sub>
 +
|<partinfo>BBa_R0062</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|RBS
 +
|<partinfo>BBa_B0034</partinfo>
 +
|SpeI+PstI
 +
|1
 +
| -
 +
|-
 +
|pSB1A3
 +
|<partinfo>pSB1A3</partinfo>
 +
|EcoRI+PstI+DpnI
 +
|3
 +
| -
 +
|-
 +
|plld
 +
| -
 +
|EcoRI+SpeI
 +
|4
 +
|433
 +
|-
 +
|}
 +
 +
The parts lysis <partinfo>BBa_K112808</partinfo>, YFP+term (<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>), <partinfo>pSB1A3</partinfo> and plld will be assembled using the 3A assembly, so made double cutting mix with these parts.
 +
 +
Gel electrophoresis was done with luxI+term (<partinfo>Bba_C0061</partinfo>, <partinfo>BBa_B0015</partinfo>), lysis <partinfo>BBa_K112808</partinfo>, YFP+term (<partinfo>BBa_E0030</partinfo>, <partinfo>BBa_B0015</partinfo>), LuxI <partinfo>Bba_C0061</partinfo> and plld.
 +
 +
P<sub>lld</sub>+lysis and pSB1A3 cut yesterday with E+P was investigated on gel. Two fragments were obtained for P<sub>lld</sub>+lysis, but the sizes of the fragments were not as expected. Since we are not sure which fragment is which, both of the fragments were cut out of the gel and purified using the QIAquick gel extraction kit. In case one of them is uncut plasmid, the unligated DNA from both bonds will be transformed tomorrow, and we will assume that if the cells transformed with DNA from one of the samples won't grow, that sample will contain digested plasmid. This sample will be ligated with pSB1A3, plated out on a ampicillin plate, and then, several cell cultures from this plate will be plated out on a chloramphenicol plate. The colonies that won't grow on chloramphenicol, is assumed to contain the correct plasmid.
 +
 +
Vgb+RBS religated, Vgb+RBS+LuxR+DTT #1, Vgb+RBS+LuxR+DTT #2, LuxI+DTT, K+RBS+LacI+DTT and his-tagged LldR was miniprepped using the Promega SV miniprep kit. The concentrations of isolated plasmid are given below:
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Plasmid
 +
!Concentration [ng/µl]
 +
|-
 +
|Vgb+RBS religated
 +
|27.3
 +
|-
 +
|Vgb+RBS+LuxR+DTT #1
 +
|26.2
 +
|-
 +
|Vgb+RBS+LuxR+DTT #2
 +
|31.7
 +
|-
 +
|LuxI+DTT
 +
|35.3
 +
|-
 +
|K+RBS+LacI+DTT
 +
|7.9
 +
|-
 +
|his-LldR
 +
|51.0
 +
|}
 +
 +
 +
===Wednesday 08.08.12===
 +
----
 +
 +
Tranformed Vgb+RBS+YFP+term (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) and Vhb+RBS+YFP+term (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) in competent DH5ɑ cells as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol]. The plasmid in both of the constructs is <partinfo>psB1A2</partinfo>. They were left in the incubator over night.
 +
 +
Inspected the petri dishes with plld+RBS (lactate induced promoter and <partinfo>BBa_B0030</partinfo>) and the religation of RBS <partinfo>BBa_B0030</partinfo>, had many more colonies on the plld+RBS than the religation, which is good. Transferred a colony of the plld+RBS (lactate promoter and <partinfo>BBa_B0030</partinfo>) and a colony of RBS <partinfo>BBa_B0030</partinfo> to liquid media, with ampicillin resistance.
 +
 +
Cut LuxI+term (<partinfo>Bba_C0061</partinfo> and <partinfo>BBa_B0015</partinfo>) and LuxR+term (<partinfo>BBa_C0062</partinfo> and <partinfo>BBa_B0015</partinfo>) with XbaI and PstI, so that they can be used as inserts. Used the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocol] with NEBuffer 3.
 +
 +
Did a gel electrophoresis with LuxI+term (<partinfo>Bba_C0061</partinfo> and <partinfo>BBa_B0015</partinfo>) and LuxR+term (<partinfo>BBa_C0062</partinfo> and <partinfo>BBa_B0015</partinfo>).
 +
 +
Purified RBS and LuxR+DTT. Resulting concentrations were 4.8 and 11.2 ng/µl, respectively.
 +
 +
===Tuesday 07.08.12===
 +
----
 +
 +
Inspected the petri dishes from yesterday, LacI <partinfo>BBa_C0012</partinfo> + terminator <partinfo>BBa_B0015</partinfo> showed colonies, the religation of the terminator <partinfo>BBa_B0015</partinfo> did not. Transferred a LacI <partinfo>BBa_C0012</partinfo> + terminator <partinfo>BBa_B0015</partinfo> colony to LB with ampicillin and inoculated at 37C with shaking.
 +
 +
Did a gel electrophoresis on plld, cut it out and extracted it, according to [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocols]. The concentration after gel extraction was: c<sub>plld</sub> = 10,6 ng/µl.
 +
 +
[[File:Plld, cut E+S, 07.08.12exp.png‎|thumb|center|400px|Picture of gel electrophoresis with plld. It was found at approximately 340 bp, as expected (344 bp).]]
 +
 +
Ligation was performed with plld as insert and RBS <partinfo>BBa_B0034</partinfo> as backbone. That means that the construct has a <partinfo>psB1A2</partinfo> plasmid. Also did a religation of the RBS <partinfo>BBa_B0034</partinfo> without any insert.
 +
 +
Transformed plld and RBS <partinfo>BBa_B0034</partinfo> in competent DH5ɑ cells, as well as the religation of RBS <partinfo>BBa_B0034</partinfo>. They were transferred to petri dishes with ampicillin resistance and left in the incubator over night.
 +
 +
Miniprepped VHB+RBS+lysis (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_K112808</partinfo>), VGB+RBS+lysis (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_K112808</partinfo>), pllD+lysis (lactate promoter and <partinfo>BBa_K112808</partinfo>) and three parallels of pBad+YFP (<partinfo>Bba_K206000</partinfo> and <partinfo>BBa_E0030</partinfo>)
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Sample
 +
!Concentration [ng/µl]
 +
|-
 +
|VHB+RBS+lysis
 +
|38,2
 +
|-
 +
|VGB+RBS+lysis
 +
|50,5
 +
|-
 +
|pllD+lysis
 +
|40,2
 +
|-
 +
|pBad+YFP #1
 +
|46,6
 +
|-
 +
|pBad+YFP #2
 +
|40,5
 +
|-
 +
|pBad+YFP #3
 +
|38,9
 +
|}
 +
 +
===Monday 06.08.12===
 +
----
 +
 +
In order to start making the construct for the biobrick we are going to improve, the parts needed for the construct were cut using the restriction digest protocol [https://2012.igem.org/Team:NTNU_Trondheim/Protocols].
 +
 +
RBS <partinfo>BBa_B0030</partinfo> will be used as a backbone and was cut with EcoRI and XbaI. LacI <partinfo>BBa_C0012</partinfo> will be an insert, and was cut with EcoRI and SpeI. The terminator <partinfo>BBa_B0014</partinfo> will be used as a backbone and was cut with EcoRI and XbaI. In addition we will test the construct using a constitutive promoter <partinfo>BBa_J23119</partinfo> which was cut using EcoRI and SpeI.
 +
 +
The inserts LacI <partinfo>BBa_C0012</partinfo> and constitutive promoter <partinfo>BBa_J23119</partinfo> were run on gel after the restriction digest.  The problem with the constitutive promoter is that it is only 35 b.p long, and is therefore difficult to use as an insert. We tried to stop the gel early to see if we could detect the constitutive promoter, but we could not see anything at all. We therefore ran the gel further and cut the LacI <partinfo>BBa_C0012</partinfo> out of the gel.
 +
 +
[[File:LacI-E+S_K-E+X_06.08.12_(1).png‎|thumb|center|400px|Gel picture of the two inserts ran on gel. Used two different ladders. To the left we used a 1kb ladder to identify the Lac I insert, and to the right we used a 50 kb ladder to identify the constitutive promoter. Expected the LacI band to be approximately 1100 bp which seems to be about right. The constitutive promoter was expected to be 35 bp, but was not detected on the gel at all.]]
 +
 +
Used the PCR purification kit and protocol from www.qiagen.com [http://www.google.no/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CFUQFjAB&url=http%3A%2F%2Fwww.qiagen.com%2FHB%2FQIAquickGelExtractionKit_EN&ei=RhYhUPiCA4iL4gS64oD4CA&usg=AFQjCNFwed-RxSRRgTFSJgtn7Fl5qisiWw&sig2=-pxyzkbLpVKt4DLlcSzQcw], on RBS <partinfo>BBa_B0030</partinfo>, pBad <partinfo>Bba_K206000</partinfo> and terminator <partinfo>BBa_B0014</partinfo>. Did a gel extraction using the Gel Extraction kit and [https://2012.igem.org/Team:NTNU_Trondheim/Protocols Protocol] on LacI <partinfo>BBa_C0012</partinfo>.
 +
 +
Did a ligation with LacI <partinfo>BBa_C0012</partinfo> and double terminator <partinfo>BBa_B0015</partinfo>, where the double terminator <partinfo>BBa_B0015</partinfo> was used as a backbone. That means, the construct has a <partinfo>psB1AK3</partinfo> backbone and resistance Ampicillin and Kanamycin. A religation of the backbone <partinfo>BBa_B0015</partinfo> was also performed.
 +
 +
The ligated LacI <partinfo>BBa_C0012</partinfo> and double terminator <partinfo>BBa_B0015</partinfo> was transformed in competent DH5ɑ cells, along with the religation of the backbone <partinfo>BBa_B0015</partinfo>. The petri dishes are left to inoculate through the night.
 +
 +
A new batch of LA-medium was made and used to make ampicillin petri dishes.
 +
 +
A restriction digest was done on the lld promoter, as described in the [https://2012.igem.org/Team:NTNU_Trondheim/Protocols protocols]. It was cut with EcoRI and SpeI, and will be an insert with RBS <partinfo>BBa_B0030</partinfo> as backbone. This will then make the plasmid <partinfo>pSB1A2</partinfo> the backbone.
 +
 +
===Sunday 05.08.12===
 +
----
 +
 +
Plasmid DNA was isolated from pllD-1B and <partinfo>BBa_B0014</partinfo> cultures were isolated by miniprep. DNA concentrations were measured as 41,2/42,5 and 53,3/52,8 ng/uL respectively (two measurements per sample).
 +
 +
Inspected agar plates in incubator inoculated on 3/8:
 +
VGB+RBS religation: 1 colony
 +
VHB+RBS+lysis: Good growth
 +
VHB+RBS religation: ~ 20 colonies
 +
VHB+RBS + lysis: Good growth
 +
pBAD (w/ backbone), religated: 40+ colonies
 +
pBAD +YFP +DTT: 7 colonies
 +
 +
Comments: Judging from the growth on the religation plates, colonies with non-insert plasmids is a possibility on all plates. pBAD is especially worrisome, as the number of colonies is lower on the insert ligation plate than on the backbone religation plate. The lysis part used in the above constructs already has an RBS, so the RBS part is redundant.
 +
 +
Induced one 5 mL culture (induction culture) pllD + lysis (3/8) with 210 uL ~ 1M lactate and addded 210 uL dH2O to another (control culture). Sampled 200 uL from both before induction, for OD measurements.
 +
 +
Inoculated 5 5 mL LB + Amp cultures with colonies from the following agar plates:
 +
VGB+RBS+Lysis (3/8)
 +
VHB+RBS+Lysis (3/8)
 +
pBAD + YFP +DTT (3/8) (3 colonies)
 +
 +
Inoculated 3 5 mL LB + Cm cultures from pllD + lysis agar plate.
 +
 +
===Friday 03.08.12===
 +
----
 +
 +
Isolated plasmid DNA from the cultures of RBS* (<partinfo>BBa_B0030</partinfo>) and LacI (<partinfo>BBa_C0012</partinfo>) inoculated yesterday.
 +
 +
Ligated pBad (<partinfo>Bba_K206000</partinfo>) together with YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), and VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>) and VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) with Lysis (<partinfo>BBa_K124017</partinfo>). These were then transformed into competent E. coli DH5α cells and plated out on petri dishes with Ampicillin.
 +
 +
Terminator <partinfo>BBa_B0014</partinfo> and re-transformed pllD in <partinfo>pSB1C3</partinfo> ("pllD 1-B") were transferred to liquid culture.
 +
 +
Ran gel on the PCR-products from yesterday.
 +
 +
Inoculated 2 x 5 mL, 1 x 10 mL and 1 x 25 mL LB + Cm with plld + lysis.
 +
 +
===Thursday 02.08.12===
 +
----
 +
 +
5 µl of the PCR product from yesterday was run on gel, but it did not give any results. We looked at the primers and thought we had done something wrong, but we did not. Will try to do the PCR one more time, but with a lower annealing temperature (55°C).
 +
 +
The constructs containing plld ligated with the lysis device(<partinfo>BBa_K112808</partinfo>), and plld in plasmid <partinfo>PSB1C3</partinfo> were miniprepped. The concentration were measured using nano drop.
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Sample
 +
!Concentration [ng/µl]
 +
|-
 +
|pllD + lysis
 +
|45,5
 +
|-
 +
|pllD in PSB1C3
 +
|42,3
 +
|}
 +
 +
Inoculated 1 colony from each of three plates containing LacI (<partinfo>BBa_C0012</partinfo>), RBS* <partinfo>BBa_B0030</partinfo> and pllD + lysis (all inoculated 1/8), into 5 mL LB + Amp (BBa_B0030, LacI)/ LB + Cm (pllD + lysis).
 +
 +
Transformed the part <partinfo>BBa_B0014</partinfo> from the distribution kit, and re-transformed pllD in pSB1C3 with DNA taken from the sample shipped to the RHIT team yesterday.
 +
 +
Reviewed the data from the oxygen-promoter experiment on monday. In conclusion, it appears that oxygen levels in the induced (anaerobic) cultures were not as low as desired, as growth rates were very similar between the aerobic and anaerobic cultures, while one would expect anaerobic cultures to grow slower. After correcting for OD, the fluorescence in the cultures at the end of the experiment was lower than the fluorescence measured from a control culture with no expected production of fluorescent protein. For this reason, a full analysis of the rest of the data was not performed.
 +
 +
===Wednesday 01.08.12===
 +
----
 +
 +
Gel purification was performed on the gel piece from yesterday, the lysis device cut with X+P. The concentration is 10,1 ng/µl. The lysis device and plld were ligated together, plld (with <partinfo>pSB1C3</partinfo>) as backbone.
 +
 +
The ligated lysis and plld were transformed into competent ''E.coli'' DH5α cells. This was also done for the biobricks lacI repressor from E. coli (<partinfo>BBa_C0012</partinfo>) and RBS (<partinfo>BBa_B0030</partinfo>).
 +
 +
The petri dishes from yesterday showed colonies on the dishes with the pSB1C3+Colisin construct (<partinfo>pSB1C3</partinfo> and <partinfo>BBa_K150009</partinfo>) and the K+RBS+Colisin construct (<partinfo>BBa_K081005</partinfo> and <partinfo>BBa_K150009</partinfo>). One colony from each petri dish were transferred to liquid medium.
 +
 +
Sent a sample of the E. coli pllD promoter in <partinfo>pSB1C3</partinfo> backbone to the iGEM team at RHIT.
 +
 +
PCR was performed on pBad (<partinfo>Bba_K206000</partinfo>) and DTT (<partinfo>BBa_B0015</partinfo>), the primers used, PCR-mix and programmed times are listed below.
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Primer
 +
!Type
 +
!Sequence
 +
!Tm [°C]
 +
|-
 +
|DTT fwd
 +
|Forward
 +
|CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAG
 +
|72
 +
|-
 +
|DTT rev
 +
|Reverse
 +
|CTCTAGAAGCGGCCGCGAATTCCAGAAATC
 +
|72
 +
|-
 +
|pBad scar fwd
 +
|Forward
 +
|TACTAGAGTACTAGTAGCGGCCGCTGCAGT
 +
|72
 +
|-
 +
|pBad fwd
 +
|Forward
 +
|TACTAGTAGCGGCCGCTGCAGTC
 +
|70
 +
|-
 +
|pBad rev
 +
|Reverse
 +
|GCTAGCCCAAAAAAACGGTATGGAGAAACAGTAGAGAG
 +
|72
 +
|}
 +
 +
Mix 1:
 +
* 1 µl dNTP
 +
* 2,5 µl 1:10 fwd primer
 +
* 2,5 µl 1:10 rev primer
 +
* 1 µl template DNA
 +
* 18 µl dH<sub>2</sub>O
 +
 +
Mix 2:
 +
* 19,25 µl d<sub>2</sub>O
 +
* 5 µl buffer with MgCl<sub>2</sub>
 +
* 0,75 µl Expand High Fidelity enzyme
 +
 +
This PCR mix was found here: http://francois.schweisguth.free.fr/protocols/High_fildelity_roche.pdf
 +
 +
Thermal timetables:
 +
 +
{|
 +
| style="padding-right:7px;" |
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+pBAD
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
 +
|1
 +
|Heated lid:
 +
|103°C
 +
|
 +
|-
 +
|2
 +
|Initial denaturation;
 +
|94°C
 +
|2 min
 +
|-
 +
|3
 +
|Denaturation;
 +
|94°C
 +
|15 s
 +
|-
 +
|4
 +
|Annealing;
 +
|66°C
 +
|30 s
 +
|-
 +
|5
 +
|Elongation;
 +
|72°C
 +
|2 min
 +
|-
 +
|6
 +
|Go to step 3, repeat 10 x
 +
|-
 +
|7
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|8
 +
|Elongation;
 +
|72°C
 +
|31 s
 +
|-
 +
|9
 +
|Go to step 7, repeat 15 x, with 5 s extra each time.
 +
|-
 +
|10
 +
|Final Elongation;
 +
|72°C
 +
|7 min
 +
|-
 +
|11
 +
|Hold
 +
|4°C
 +
|∞
 +
|}
 +
 +
| style="padding-left:7px;" |
 +
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+ DTT
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
 +
|1
 +
|Heated lid:
 +
|103°C
 +
|
 +
|-
 +
|2
 +
|Initial denaturation;
 +
|94°C
 +
|2 min
 +
|-
 +
|3
 +
|Denaturation;
 +
|94°C
 +
|15 s
 +
|-
 +
|4
 +
|Annealing;
 +
|66°C
 +
|30 s
 +
|-
 +
|5
 +
|Elongation;
 +
|72°C
 +
|3 min
 +
|-
 +
|6
 +
|Go to step 3, repeat 10 x
 +
|-
 +
|7
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|8
 +
|Elongation;
 +
|72°C
 +
|31 s
 +
|-
 +
|9
 +
|Go to step 7, repeat 15 x, with 5 s extra each time.
 +
|-
 +
|10
 +
|Final Elongation;
 +
|72°C
 +
|7 min
 +
|-
 +
|11
 +
|Hold
 +
|4°C
 +
|∞
 +
|}
 +
|}
 +
 +
===Tuesday 31.07.12===
 +
----
 +
 +
The restriction digest for colicin cut with E+P made yesterday was purified using the QIAquick PCR purification kit, and after purification, the following ligations were made:
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Insert
 +
!Backbone
 +
!Volume of insert [µl]
 +
!Volume of backbone [µl]
 +
!Amount of T4 DNA ligase buffer [µl]
 +
!Amount of T4 DNA ligase [µl]
 +
!Amount of dH<sub>2</sub>O added [µl]
 +
|-
 +
|Colicin cut with E+P
 +
|pSB1C3 cut with E+P
 +
|4.0
 +
|2.0
 +
|1.0
 +
|0.5
 +
|2.5
 +
|-
 +
|Colicin cut with X+P
 +
|K+RBS cut with S+P
 +
|7.0
 +
|5.0
 +
|1.5
 +
|0.75
 +
|0.75
 +
|-
 +
| -
 +
|pSB1C3 cut with E+P
 +
| -
 +
|2.0
 +
|1.0
 +
|0.5
 +
|6.5
 +
|-
 +
| -
 +
|K+RBS cut with S+P
 +
| -
 +
|5.0
 +
|1.5
 +
|0.75
 +
|7.75
 +
|}
 +
 +
Both the ligation and the religations were transformed to competent ''E.coli'' DH5α cells.
 +
 +
Restriction digest was run on the Lysis device (<partinfo>BBa_K112808</partinfo>) and the lld-promoter, as described in the [[Team:NTNU_Trondheim/Protocols|protocol]]. Lysis was cut with X and P, plld cut with S and P. Plld was purified using a PCR purification kit, gel electrophoresis was done with the lysis device. The concentration of the plld was 3,2 ng/µl.
 +
 +
===Monday 30.07.12===
 +
----
 +
 +
Today, we started testing the Vgb (<partinfo>BBa_K561001</partinfo>) and Vhb (<partinfo>BBa_K258005</partinfo>) promoters. For both promoters, we have made test constructs consisting of promoter, RBS (<partinfo>BBa_B0034</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and terminator (<partinfo>BBa_B0015</partinfo>). The testing was carried out by using two parallel cultures of each promoter construct, and inducing one of the parallels by bubbling the culture with nitrogen gas to remove oxygen. Samples were collected every 10 minutes.
 +
 +
Further, the constitutive promoter containing RBS (<partinfo>BBa_K081005</partinfo>) was cut with S+P, and the purified colicin PCR product were cut with X+P, to be ligated tomorrow. Another colicin PCR product sample was cut with E+P, to be ligated into the already cut pSB1C3 tomorrow.
 +
 +
We also test cutted P<sub>lld</sub> with BpmI and AflIII. This was performed because the number of religations for the pSB1C3 plasmid was higher than it should have been. One of the samples to be test cutted was taken from the miniprepped plasmids of one of the colonies on the religation plate, to be used as negative controll, and three parallel samples from the ligation plate were test cutted. The gel pictures are shown below:
 +
 +
{| style="text-align:center;"
 +
|[[File:AflIII-testkutt_Plld_i_pSB1C3_30.07.12.png|thumb|center|450px| Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with AflIII. AflIII cuts both inside the promoter region, and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 277 bp, and one on 2136. All parallels from the ligation shows two bonds at the expected positions.]]
 +
|[[File:BpmI-testkutt_Plld_i_pSB1C3_30.07.12.png|thumb|center‎|450px|Religated pSB1C3, and three parallels of ligated P<sub>lld</sub> and pSB1C3 investigated by cutting with BpmI. BpmI also cuts both inside the promoter region and in the backbone, so if the promoter is present in the plasmid, the expected result for this test cut is two fragments; one on 898 bp, and one on 1515. Only one band is present on the gel.]]
 +
|}
 +
 +
The results from the test cutting were ambigous. The test cut performed with AflIII show the expected fragments, while the test cut performed with BpmI do not. It seems that the promoter is there in all thre parallels; if not it would have been impossible to yield two fragements in the AflIII test cut. But still, if the promoter sequence is as it should have been, also the test cut with BpmI should have yielded two fragments. It is, however, possible, that the BpmI restriction site inside the promoter is ok, but that the restriction site in the plasmid is damaged.
 +
 +
===Sunday 29.07.12===
 +
----
 +
 +
Made LB medium in preparation for experiment with the oxygen-sensitive promoters tomorrow.
 +
Inspeccted the <partinfo>pSB1C3</partinfo> religation plate in the incubator. Many colonies that were not visible at the time of the colony count yesterday has now appeared. The religation and the pllD plate now contains about the same number of colonies, casting doubt on the sucess of the transformation. Moved the plate to the fridge.
 +
 +
Extracted plasmid DNA by miniprep from the pllD (unconfirmed) and <partinfo>pSB1C3</partinfo> religation cultures inoculated yesterday, and the culture of <partinfo>BBa_E0040</partinfo> (A. vic. GFP) inoculated 26/7. DNA concentrations were measured with NanoDrop, as follows (two parallel measurements were made for each sample):
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Sample
 +
!Concentration [ng/µl]
 +
|-
 +
|pllD 1
 +
|24,0/22,2
 +
|-
 +
|pllD 2
 +
|25,9/26,5
 +
|-
 +
|pllD 3
 +
|22,8/23,9
 +
|-
 +
|<partinfo>pSB1C3</partinfo> 1
 +
|15,4/15,5
 +
|-
 +
|<partinfo>pSB1C3</partinfo> 2
 +
|20,7/20,7
 +
|-
 +
|<partinfo>BBa_E0040</partinfo>
 +
|38,2/37,8
 +
|}
 +
 +
DNA from all the above samples except GFP was digested with Xba1 and Spe1. The digested samples were analyzed by gel electrophoresis to find out whether the desired insert was obtained in any of the samples. 75 V was applied for 45 min, and the gel was imaged. After imaging, electrophoresis was continued at 40 V for a further 30 minutes before imaging again. The result of the second imaging is shown below.
 +
 +
[[file:ChemiDoc 2012-07-29 22hr 41min pllD pSB1C3.jpg|thumb|450px|center]]
 +
 +
 +
Results of gel electrophoresis: All three samples from the pllD plate showed a short band identical between these samples, but it is difficult to determine the exact size - there seems to be an inconsistency in the migration of the 50 bp ladder on the left side of the gel and the 1 kb ladder on the right. Looking at the 50 bp ladder, the short band in the three first samples seems to be between the third and the fourth band from the bottom, representing 150 and 200 bp respectively. However, looking at the 1 kb ladder on the right, the short bands lie between the first and second ladder bands, equaling 250 and 500 bp respectively. The position of the short bands with respect to the 1 kb ladder appears to be consistent with the expected size of the pllD insert.
 +
 +
Taken together, it seems possible that the short band represents the desired insert, but the samples should be analyzed again to get more data. The samples from the <partinfo>pSB1C3</partinfo> religation plate does not show this band and is overall much fainter.
 +
 +
The top bands in the pllD wells are on heigth with the seventh band from the bottom, equaling 2500 bp, in the Generuler 1kb ladder on the right. This is approximately the size of the <partinfo>pSB1C3</partinfo> plasmid and the pllD insert together. The two wells on the right containing DNA from the <partinfo>pSB1C3</partinfo> religation plate show two faint bands each: One slightly above the top bands in the pllD wells, and one shorter band slightly above the 1000 bp reference band in the 1 kb ladder.
 +
 +
===Saturday 28.07.12===
 +
----
 +
 +
Inspected the plates with PllD in <partinfo>pSB1C3</partinfo> and <partinfo>pSB1C3</partinfo> religation transformants inoculated yesterday. Growth on both plates, but more on the PllD plate. No growth on negative control (non-transformed cells). Approximate colony cound:
 +
Religation: 73
 +
pllD: 240
 +
 +
Assuming other factors are equal, based on these numbers a colony on the lldP plate should have about a 2/3 chance of containing the desired insert. Suggested course of action: Inoculate several colonies from the lddp plate, miniprep, perform a test restriction digest and gel electrophoresis to check size.  Use colonies from the religation plate for reference: Plasmids with the desired insert should be about 344 bp larger than the religated plasmid without insert.
 +
 +
Inoculated 3 colonies from the PllD plate and 2 colonies from the <partinfo>pSB1C3</partinfo> religation plate into 5 mL LB + Cm, and placed the cultures in 37 C shaking incubator. Placed the plates back in 37 C cabinet.
 +
 +
Updated information about stored DNA samples and glycerol stocks on the wiki.
 +
 +
Evening: Moved lldP plate from incubator to fridge.
 +
 +
===Friday 27.07.12===
 +
----
 +
 +
The PCR batches run yesterday were investigated on gel electrophoresis, and the gel picture shows that colicin has been made!
 +
 +
[[File:Plld,_LldR,_kolisin_PCR-produkt_26.07.12_exp.jpg|thumb|center|450px|PCR batches for P<sub>lld</sub>, LldR, and colicin investigated on gel. Apparently, no PCR product was obtained for P<sub>lld</sub> and LldR, but the colicin sample loaded in the third well shows a bond above 2000 bp. It is hard to imagine that this can be anything else than colicin.]]
 +
 +
The colicin PCR product were purified using the QIAquick PCR Purification Kit, and the concentration of product was measured to be 15.3 ng/µl.
 +
 +
Linearized pSB1C3 plasmid was digested with EcoRI and PstI, according to the procedure for [http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones digestion of linearized plasmids], and ligated with the lld promoter made by PCR on wednesday. Religation was also performed, and both the ligated plasmid and the religated plasmid were transformed to competent ''E.coli'' DH5α cells.
 +
 +
New primers were ordered for LldR, this time containing the biobrick prefix and suffix.
 +
 +
Discussed experimental design for testing of oxygen-sensitive promoters with Martin. Made a plan for testing both promoters on Monday.
 +
 +
Measured OD600 of samples from the cultures used to make glycerol stocks yesterday. The samples were taken yesterday and kept at 5 C until today. Results:
 +
EYFP (RBS+ LVA- TERM)(<partinfo>BBa_E0430</partinfo>): 1,00
 +
pTet RFP (<partinfo>BBa_I13521</partinfo>) : 2,14
 +
ptet GFP (<partinfo>BBa_I13522</partinfo>) : 0,86
 +
 +
Measured YFP, GFP and RFP fluorescence from the following cultures ([https://www.dropbox.com/s/ku0l7enk5c6trkz/Fluorescence%20measurements%2027.07.xlsx data]):
 +
LB (22/7) (D1)
 +
K + RBS + YFP + DTT (19/7) (D2)
 +
ptet mRFP (23/7) (D3)
 +
ptet mRFP (26/7) (D4)
 +
ptet GFP (26/7) (D5)
 +
EYFP (RBS+ LVA- TERM) (26/7) (D6)
 +
 +
Measurements confirmed that ptet GFP and ptet MRFP produce the desired products and can be used as positive controls. Significant overlap was observed between GFP and YFP fluorescence.
 +
 +
===Thursday 26.07.12===
 +
----
 +
 +
The PCR-products for P<sub>lld</sub>, LldR, and colicin were run on gel:
 +
 +
 +
[[File:Plld,_LldR,_kolisin_PCR-produkt_25.07.12_exp.png|thumb|center|450px|The obtained PCR products from the three PCRs run wednesday. The first well after the ladder contains the PCR product for the P<sub>lld</sub> promoter, which in theory should have been 344 bp long. One PCR product was obtained, but even if it is closer to the lowest band on the ladder, being 250 bp long, it is assumed that the correct product was obtained. The second well after the ladder apparently contains no products, but should have contained LldR, being 777 bp long. The third well after the ladder should have contained colicin, being 2050 bp long. In this well, it is possible to see a weak band close to the 3000 band on the ladder, but whether or not this is the correct product is not certain.]]
 +
 +
 +
Since we are assuming that the P<sub>lld</sub> promoter is correct, this PCR product were purified using the QIAquick PCR purification kit. The concentration after purification was 16.5 ng/µl DNA.
 +
The biobrick were then cut with EcoRI and PstI, to be ligated with EcoRI and PstI digested pSB1C3 tomorrow.
 +
 +
As no PCR product was made for LldR, and none of the expected product was made in the case of colicin, a new PCR were run, also including P<sub>lld</sub>, to investigate it on gel again, to see if the length of it relative to the ladder is more precise. For the case of the new PCR mixes, 50 µl were made instead of 20 µl, and 1 µl DMSO, which is known to increase the yield of desired product, was added. The PCR mixes were made according to the following procedure:
 +
 +
* 31.5 µl dH<sub>2</sub>O
 +
* 10 µl 5x Phusion buffer
 +
* 1 µl dNTPs
 +
* 2.5 µl fwd primer (both primers having concentration 10 µM)
 +
* 2.5 µl rev primer
 +
* 1 µl template DNA
 +
* 1 µl DMSO
 +
* 0.5 µl Finnzymes Phusion HF DNA polymerase
 +
 +
The thermocyclers were programmed to use the same PCR programs as the ones used wednesday.
 +
 +
 +
GFP (<partinfo>BBa_E0040</partinfo>) were also transferred to liquid culture.
 +
 +
Today, we also had a video conference with the [https://2012.igem.org/Team:RHIT Rose Hulman Institute of Technology iGEM team], discussing how we could collaborate with each other. It was really nice meeting you, and hopefully, we'll be seing you in Boston in November:-)
 +
 +
Inspected plates with EYFP + LVA and ptet GFP inoculated 24/7, incubated overnight and kept in fridge since yesterday. Good growth.
 +
 +
Discarded K + RBS + RFP plate (17/7) found in incubator cabinet.
 +
 +
Made glycerol stocks from the following cultures:
 +
EYFP + LVA (<partinfo>BBa_E0430</partinfo>) (26/7)
 +
ptet GFP (<partinfo>BBa_I13522</partinfo>) (26/7)
 +
ptet mRFP (<partinfo>BBa_I13521</partinfo>) (26/7)
 +
 +
Re-inoculated following liquid cultures in new LB + amp medium (50 uL to 5 mL), followed by miniprep of the old culture.
 +
VGB + RBS + YFP + DTT (25/7)
 +
VHB + RBS + YFP + DTT (25/7)
 +
EYFP + LVA (25/7)
 +
ptet GFP (25/7)
 +
 +
 +
Measured DNA concentrations (two parallels):
 +
VGB + RBS + YFP + DTT: 23,2/22,6
 +
VHB + RBS + YFP + DTT: 15,7/15,5
 +
EYFP + LVA: 19,5/20,1
 +
ptet GFP: 23,2/22,3
 +
 +
Ran gel electrophoresis of the latest PCR products (plld, LldR, colicin operon).
 +
 +
===Wednesday 25.07.12===
 +
----
 +
 +
The pBad+RBS+YFP+DTT, the Vgb+RBS+YFP+DTT and the Vhb+RBS+YFP+DTT constructs were transferred to liquid culture. The primers we ordered two and a half weeks ago (!) also arrived this morning, so both the lld promoter, LldR and colicin were amplified using PCR. The following PCR mix was used for the three samples:
 +
 +
* 12.4 µl dH<sub>2</sub>O
 +
* 4 µl 5x Phusion buffer
 +
* 0.4 µl dNTPs
 +
* 1 µl fwd primer (both primers having concentration 10 µM)
 +
* 1 µl rev primer
 +
* 1 µl template DNA
 +
* 0.2 µl Phusion HF DNA polymerase
 +
 +
In the case of both the lld promoter and the LldR gene, the genome of ''E.coli'' K12 (strain ER2925) was used as template. For the colicin batch, the <partinfo>BBa_K150009</partinfo> biobrick was used as template.
 +
Two of the primer sets contained prefix and suffix, and for these, two different melting temperatures were determined. The reason for this is that in the first cycles, the only the primer parts able to bind to the template will be determining for the melting temperature, while in the later cycles, where the amplified DNA is dominating as template, also the prefix / suffix parts of the primers will be able to bind to the DNA, and hence the primers will obtain higher melting temperatures. For the two PCR mixes containing primers with prefix and suffix, a three step protocol was used in the first ten cycles, while a two step protocol was used in the last fifteen cycles. Phusion High Fidelity DNA Polymerase (Finnzymes). To calculate the melting temperature, we used Finnzymes' Tm calculator (http://www.finnzymes.fi/tm_determination.html).
 +
 +
{| class="wikitable" style="text-align:centre;margin: 1em auto 1em auto;"
 +
!Primer
 +
!Type
 +
!Sequence
 +
!Tm [°C]
 +
|-
 +
|lld promoter fwd
 +
|Forward
 +
|cacattcctataggccgagtaaggt
 +
|66.2
 +
|-
 +
|lld promoter rev
 +
|Reverse
 +
|gcaggtctcctggagtccacgc
 +
|73.8
 +
|-
 +
|lldR fwd
 +
|Forward
 +
|atgattgttttacccagacgcctgt
 +
|69.3
 +
|-
 +
|lldR rev
 +
|Reverse
 +
|tcatgcgtttttctccctcgaat
 +
|69.7
 +
|-
 +
|Colicin fwd
 +
|Forward
 +
|atggaaaccgcggtagcgta
 +
|68.7
 +
|-
 +
|Colicin rev
 +
|Reverse
 +
|tgcgatggtccctccctgaa
 +
|72.6
 +
|}
 +
 +
The following PCR programs were used:
 +
 +
{|style="text-align:center; margin: 1em auto 1em auto;" |
 +
 +
| style="padding-right:10px"; |
 +
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+ lld promoter
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
 +
|1
 +
|Heated lid:
 +
|103°C
 +
|
 +
|-
 +
|2
 +
|Initial denaturation;
 +
|98°C
 +
|30 s
 +
|-
 +
|3
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|4
 +
|Annealing;
 +
|69°C
 +
|10 s
 +
|-
 +
|5
 +
|Elongation;
 +
|72°C
 +
|6 s
 +
|-
 +
|6
 +
|Go to step 3, repeat 10 x
 +
|
 +
|
 +
|-
 +
|7
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|8
 +
|Elongation;
 +
|72°C
 +
|6 s
 +
|-
 +
|9
 +
|Go to step 7, repeat 15 x
 +
|
 +
|
 +
|-
 +
|10
 +
|Final elongation;
 +
|72°C
 +
|10 min
 +
|-
 +
|11
 +
|Hold
 +
|4°C
 +
|∞
 +
|}
 +
 +
| style="padding-left:10px;" |
 +
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+ Colicin
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
 +
|1
 +
|Heated lid:
 +
|103°C
 +
|
 +
|-
 +
|2
 +
|Initial denaturation;
 +
|98°C
 +
|30 s
 +
|-
 +
|3
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|4
 +
|Annealing;
 +
|70°C
 +
|10 s
 +
|-
 +
|5
 +
|Elongation;
 +
|72°C
 +
|31 s
 +
|-
 +
|6
 +
|Go to step 3, repeat 10 x
 +
|
 +
|
 +
|-
 +
|7
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|8
 +
|Elongation;
 +
|72°C
 +
|31 s
 +
|-
 +
|9
 +
|Go to step 7, repeat 15 x
 +
|
 +
|
 +
|-
 +
|10
 +
|Final elongation;
 +
|72°C
 +
|10 min
 +
|-
 +
|11
 +
|Hold
 +
|4°C
 +
|∞
 +
|}
 +
 +
|-
 +
 +
|colspan="2" |
 +
 +
{|class="wikitable" style="margin: 1em auto 1em auto;"
 +
|+ lldR
 +
! Step
 +
! Action
 +
! Temperature
 +
! Duration
 +
|-
 +
|1
 +
|Heated lid:
 +
|103°C
 +
|
 +
|-
 +
|2
 +
|Initial denaturation;
 +
|98°C
 +
|30 s
 +
|-
 +
|3
 +
|Denaturation;
 +
|98°C
 +
|10 s
 +
|-
 +
|4
 +
|Annealing;
 +
|70°C
 +
|10 s
 +
|-
 +
|5
 +
|Elongation;
 +
|72°C
 +
|24 s
 +
|-
 +
|6
 +
|Go to step 3, repeat 25 x
 +
|
 +
|
 +
|-
 +
|7 
 +
|Final elongation;
 +
|72°C
 +
|10 min
 +
|-
 +
|8
 +
|Hold
 +
|4°C
 +
|∞
 +
|}
 +
 +
|}
 +
 +
We also transformed the biobrick <partinfo>BBa_E0040</partinfo>, which is a protein coding part coding for GFP.
 +
 +
===Tuesday 24.07.12===
 +
----
 +
 +
Transformed the pBAD+RBS+YFP+DTT (<partinfo>Bba_K206000</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), VGB+RBS+YFP+DTT (<partinfo>BBa_K561001</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>), and VHB+RBS+YFP+DTT (<partinfo>BBa_K258005</partinfo>, <partinfo>BBa_B0034</partinfo>, <partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) constructs ligated yesterday to supercompetent ''E.coli'' Dh5α cells.
 +
 +
We also started making our iGEM matchmaker, which will be useful for iGEM teams wanting collaboration with other teams.
 +
 +
Some of our primers arrived today, so tomorrow the PCR fun will start.
 +
 +
Transformed the BioBrick parts <partinfo>BBa_E0430</partinfo> and <partinfo>BBa_I13522</partinfo> using the iGEM 2011 DNA distribution kit.
 +
 +
===Monday 23.07.12===
 +
----
 +
 +
We continnued the assembly of the promoter test constructs. As the test gel run on friday showed the expected fragment, the YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>) samples were separated using gel electrophoresis. The inserts were cut out of the gel, as shown below, and purified using the QIAquick Gel Extraction Kit.
 +
 +
[[File:YFP_insert_23.07.12 exp.png|thumb|center|400px|The picture is showing two parallels of the YFP+DTT sample, containing an insert to be used in ligation with promoter+RBS constructs. The inserts, shown in red boxes, were cut out of the gel and purified.]]
 +
 +
The pBad+RBS (<partinfo>Bba_K206000</partinfo>), Jen1+RBS (<partinfo>BBa_K284002</partinfo> and <partinfo>BBa_B0034</partinfo>), Vgb+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>) and Vhb+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) backbones were purified using the QIAquick PCR Purification Kit. In all of the above it is the plasmid from RBS that's the backbone (<partinfo>psB1A2</partinfo>). Concentrations of both inserts and backbones after purification are given below:
 +
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Biobrick
 +
!Restriction enzymes used in digest
 +
!Concentration [ng/µl]
 +
|-
 +
|YFP+DTT
 +
|XbaI + PstI
 +
|4.1
 +
|-
 +
|pBad+RBS
 +
|SpeI + PstI
 +
|0.5
 +
|-
 +
|Vgb+RBS
 +
|SpeI + PstI
 +
|1.7
 +
|-
 +
|Vhb+RBS
 +
|SpeI + PstI
 +
|2.4
 +
|-
 +
|Jen1+RBS
 +
|SpeI + PstI
 +
|1.2
 +
|}
 +
 +
VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>), VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) and pBAD+RBS (<partinfo>Bba_K206000</partinfo>) were all ligated with YFP+DTT (<partinfo>BBa_E0030</partinfo> and <partinfo>BBa_B0015</partinfo>).
 +
 +
For the YFP + DTT fragment, DNA concentration measurements with NanoDrop after isolation with the quiagen kit were inconsistent. For the samples made from the two gel bands, values ranged from 3.7 to 14.8 and 4.0 to 24.4 ng/uL, respectively. Conclusion: Several measurements should always be made.
 +
 +
Inspected mRFP and RBS plates from yesterday. Good growth on both. No growth on negative controlplate.
 +
 +
Inoculated both mRFP and RBS from the plates into 5 mL LB + Amp (21/7), placed in 37 C shaking incubator
 +
 +
===Sunday 22.07.12===
 +
----
 +
 +
Attempted to measure GFP fluorescence in culture inoculated yesterday with negative result.
 +
 +
Inoculated two new cultures of K + RBS and K + RBS + GFP + DTT by transferring 30 uL from the cultures inoculated yesterday to 3 mL LB + Amp (medium prepared yesterday and kept in refrigerator). Attempted to measure GFP fluorescence at aprox. 2 and 4 h inoculation (OD ~ 0.2 and ~ 1.2} with negative result.
 +
 +
Is the problem with the construct itself/expression of GFP from the plasmid, or with plasmid stability? All cultures tested for GFP fluorescence so far have been "old" (probable stationary phase) or inoculated from "old" cultures. Reports indicate that B-lactamase in old culture media may degrade ampicillin at a high rate and lead to decreased plasmid stability, also when a new culture is inoculated using medium from an existing culture. To rule out this being the problem, a fresh culture should be inoculated from an agar plate and tested for fluorescence during log phase. Another possibility is to inoculate from an existing culture by spinning down the cells, washing the pellet with fresh medium (to remove B-lactamase), decant the washing medium and resuspend the cells in new growth medium.
 +
 +
Transformed cells with the part <partinfo>BBa_I13521</partinfo> (pTet mRFP) from the distribution kit and the part <partinfo>BBa_B0034</partinfo> from miniprep DNA.
 +
 +
===Saturday 21.07.12===
 +
----
 +
Remeasured GFP and RFP fluorescence using different wavelengths and new samples. Results still negative. Re-measured YFP fluorescence on original sample retained in 96-well plate. Results were similar to initial measurement, indicating that YFP is stable for several days.
 +
 +
Inoculated two 5 mL cultures of LB with K + RBS and K + RBS + GFP + DTT respectively, from agar plates.
 +
 +
===Friday 20.07.12===
 +
----
 +
 +
Attempted to measure fluorescence of cultures inouculated yesterdat with transformants harboring GFP and RFP under constitutive promoter (<partinfo>BBa_K081005</partinfo>). Results were negative - neither GFP or RFP seems to be expressed.
 +
 +
As the GFP and RFP constructs failed, work was started on assembling the promoters Jen1, pBAD, VGB and RBS with YFP. Plasmid DNA of JEN1+ RBS, VGB + RBS, VHB + RBS and pBAD + RBS in pSB1A2 was digested with Spe1 and Pst1. Plasmid DNA of YFP + DTT in psB1AK3 was digested with XbaI and Pst1. For YFP + DTT, digestion was performed on two samples. 4 uL of each sample digested DNA was used for gel electrophoresis to confirm the result,, the rest was stored at - 20 C. Gel visualization showed the expected fragments.
 +
 +
===Thursday 19.07.12===
 +
----
 +
 +
Designed new primers for the DDT terminator in pSB1A3 and ordered all primers designed today and yesterday. Contacted GATC and asked them to send us barcode stickers necessary for sequencing. They were also asked if they would like to join us as sponsors, providing us with sequencing.
 +
 +
Transferred transformants with RFP and GFP constructs to liquid culture.
 +
 +
Measured fluorescence in liquid culture of cells transformed with YFP (<partinfo>BBa_E0030</partinfo>) under a constitutive promoter (<partinfo>BBa_K081005</partinfo>). Results indicated that YFP was expressed ([https://www.dropbox.com/s/6npz5uux53o5qhy/Constitutive%20YFP%20fluorescence%2019.06.xlsx data]). Due to  the measurement of high fluorescence in the reference LB medium at the indicated maximum emission wavelength 527 nm, emission was measured centered at 544 nm.
 +
 +
Researched the lysis device and luxR+HSL induced promoter. The activity of the lysis device <partinfo>BBa_K112808</partinfo> under the control of <partinfo>BBa_R0062</partinfo> with LuxR constitutively coexpressed under TetR (<partinfo>BBa_R0040</partinfo> has been characterized by Pasotti et al. ([http://www.jbioleng.org/content/5/1/8])
 +
 +
===Wednesday 18.07.12===
 +
----
 +
 +
Colonies from the petri dishes of VHB+RBS+RFP+DTT, VGB+RBS+GFP+DTT, Jen1+RBS+GFP+DTT and K+RBS+YFP+DTT were transferred to liquid medium. K+RBS+GFP+DTT and K+RBS+RFP+DTT didn't have any colonies, new petri dishes were made. The old and the new were incubated at 37C.
 +
 +
Inspected DTT, YFP and D2 plates. All showed good growth. Moved the plates to the frigde and discarded the old DTT; YFP and D2 plates (dates, 24/6, 20/6 and 20/6, respectively).
 +
 +
Inspectd RFP and VGB plates made yesterday. Both showed good growth. No growth on negative control plates. Discarded old VGB and RFP plates (dated 25/6 and 20/6). Prepared and stored glycerol stock of RFP and VGB.
 +
 +
Made a new wiki design. Designed primers for amplification of the lldR gene from K12 and the DDT terminator in pSB1A3.
 +
 +
===Tuesday 17.07.12===
 +
----
 +
 +
Transformed Vhb+RBS+RFP+DTT, Vgb+RBS+GFP+DTT, Jen1+RBS+GFP+DTT and K+RBS with YFP+DTT, GFP+DTT and RFP+DTT, a control was also made. Ampicillin petri dishes were used, and they are left to incubate for the night.
 +
 +
Prepared and stored glycerol stocks from liquid cultures of DTT, CFP, YFP and "D2" per our protocol.
 +
Measured OD on the cultures used. Result:
 +
DTT: 1,42<br>
 +
CFP: 1,44<br>
 +
YFP: 1,41<br>
 +
D2: 1,35<br>
 +
 +
Performed an experiment to assess effect of induction of lysis device on the number of colony forming units (CFU). Combined the two 3 mL lysis + pBAD cultures inoculated yesterday, mixed and pipetted out 2 mL to two each of two new tubes. Added 19 uL 0,1 M arabinose (solution made 10/7) to one (induced) and equal volume dH2O to the other (control). The cultures were then incubated at 37 C with shaking. Immediately before induction, and at 30 min, 1 h, 2h and 3h after induction, 1 uL culture was pipetted out from each culture, diluted in 1000 uL H2O, the dilution vortex mixed, and 1 uL of the dilution pipetted out into 1000 uL again for a 10^-6 dilution. For each culture and time point, two LA + Amp plates were inoculated with 20 uL and 200 uL of the 10^-6 dilution, respectively. Inoculated plates were incubated at 37 C.
 +
 +
OD measurements were made immediately before induction, at 30 min, 1h and 3h post-induction. Results:
 +
Pre-induction:
 +
Induced: 1,72
 +
Control: 1,81
 +
 +
30 min:
 +
Induced: 1,33
 +
Control: 1,78
 +
 +
1 h:
 +
Induced: 1,02
 +
Control: 1,79
 +
 +
3 h:
 +
Induced: 1,00/1,09 (remeasurement, new sample)
 +
Control: 2,50/2,45 (remeasurement, new sample)
===Monday 16.07.12===
===Monday 16.07.12===
Line 23: Line 1,044:
-
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 (<partinfo>BBa_K081005</partinfo>), 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.
+
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. The concentrations after purification are as follows:
 +
 
 +
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
 +
!Construct
 +
!Concentration [ng/µl]
 +
|-
 +
|YFP+DTT
 +
|4.9
 +
|-
 +
|GFP+DTT
 +
|10.6
 +
|-
 +
|RFP+DTT
 +
|32.1
 +
|-
 +
|CFP+DTT
 +
|5.3
 +
|-
 +
|K+RBS
 +
|8.2
 +
|-
 +
|Vhb+RBS
 +
|8.1
 +
|-
 +
|Vgb+RBS
 +
|9.0
 +
|-
 +
|Jen1+RBS
 +
|10.4
 +
|}
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.
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.
 +
 +
Inspected the plates made yesterday. All plates showed numerous (100++) colonies or bacterial lawn. No difference between plates inouclated with culture diluted in LB and diluted in H2O was immediately appearant. Conclusion: For making new plates from saturated E. coli cultures in LB, a volume of 20 uL from a 10 000 - 100 000 dilution in H2O is probably suitable. Discarded the plates inoculated with LB-diluted culture. Placed the plates inoculated with H2O diluted culture in the refrigerator.
 +
 +
Inoculated two 3 mL LB+Amp aliquots with 30 uL pBAD + lysis culture, placed in 37 C shaking incubator.
 +
 +
Prepared and stored glycerol stocks of LuxR+HSL, LuxR, Lysis, pBAD, GFP and pBAD+lysis.
 +
 +
Made a new stock solution (2 mL) of chloramphenicol.
 +
 +
Made new La+Amp plates (27).
 +
 +
Plated out YFP, CFP, DTT and "D2" from the cultures made 13/7. Inoculated a liquid culture of RFP from the plate made on 20/6, and pf VGB from the plate made 25/6.
 +
 +
===Sunday 15.07.12===
 +
----
 +
Removed liquid cultures of pBAD + lysis,pBAD, LuxR, LuxR+HSL regulated promoter, GFP lysis device and LuxR hybrid promoter from incubator. RFP showed no visible growth - may have used the wrong antibiotic (amp instead of kan).
 +
 +
Plated out LuxR+HSL, Lysis, pBAD, LuxR, GFP and pBAD + lysis, as follows: 1 uL of each culture was added to both of two eppendorf tubes containing 1000 uL LB medium and 1000 uL H2O, respectively. The dilutions were vortex mixed and 20 uL of each dilution plated out on plates containing LA + amp. The plates were incubated at 37 C, while the remainder of the LB dilutions were incubated at 37 C with shaking. All other liquid cultures were placed in the refrigerator.
===Saturday 14.07.12===
===Saturday 14.07.12===
Line 64: Line 1,132:
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].
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.
+
LuxI+DTT (<partinfo>Bba_C0061</partinfo> and <partinfo>BBa_B0015</partinfo>) and LuxR+DTT (<partinfo>BBa_C0062</partinfo> and <partinfo>BBa_B0015</partinfo>) 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.
We had our weekly meeting with the advisors. A very quick summary can be found in the Dropbox folder.
 +
 +
Inoculated new liquid cultures from existing agar plates of E. coli DH5a cells transformed with the following BioBrick parts/constructs, in preparation for making new plates:
 +
 +
*pBAD + lysis construct (<partinfo>Bba_K206000</partinfo> and <partinfo>BBa_K112808</partinfo>)<br>
 +
*pBAD strong promoter (<partinfo>Bba_K206000</partinfo>)
 +
*LuxR (<partinfo>BBa_C0062</partinfo>)<br>
 +
*LuxR+HSL regulated promoter (<partinfo>BBa_R0062</partinfo>)<br>
 +
*GFP (<partinfo>BBa_K082003</partinfo>)<br>
 +
*YFP (<partinfo>BBa_E0030</partinfo>)<br>
 +
*CFP (<partinfo>BBa_E0020</partinfo>)<br>
 +
*Hybrid promoter: HSL-LuxR activated, P22 C2 repressed (<partinfo>BBa_K145150</partinfo>)<br>
 +
*T4 lysis device (<partinfo>BBa_K112808</partinfo>)<br>
===Thursday 12.07.12===
===Thursday 12.07.12===
----
----
-
LuxI and LuxR were cut and ligated with the DTT terminator. LuxI+DTT and LuxR+DTT were thereafter transformed.
+
LuxI (<partinfo>Bba_C0061</partinfo>) and LuxR (<partinfo>BBa_C0062</partinfo>) were cut and ligated with the DTT terminator (<partinfo>BBa_B0015</partinfo>). 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.  
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.
+
Restriction digestion were performed on Jen1+RBS (<partinfo>BBa_K284002</partinfo> and <partinfo>BBa_B0034</partinfo>), VGB+RBS (<partinfo>BBa_K561001</partinfo> and <partinfo>BBa_B0034</partinfo>), VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) and LuxR (<partinfo>BBa_C0062</partinfo>) 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===
===Wednesday 11.07.12===
Line 97: Line 1,177:
----
----
-
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:
+
As the restriction cutting of RFP (<partinfo>Bba_E1010</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>), GFP (<partinfo>BBa_K082003</partinfo>) and CFP (<partinfo>BBa_E0020</partinfo>) 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:
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
Line 117: Line 1,197:
|}
|}
-
The two parallels of the terminator cut yesterday with E+X was purified using the QIAquick PCR purification kit. Concentrations are given below:
+
The two parallels of the terminator (<partinfo>BBa_B0015</partinfo>) cut yesterday with E+X was purified using the QIAquick PCR purification kit. Concentrations are given below:
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
Line 131: Line 1,211:
|}
|}
-
Test cutting of the fluorescent protein parts (RFP, YFP, GFP and CFP) was performed. Results are shown below:
+
Test cutting of the fluorescent protein parts (RFP (<partinfo>Bba_E1010</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>), GFP (<partinfo>BBa_K082003</partinfo>) and CFP (<partinfo>BBa_E0020</partinfo>)) was performed. Results are shown below:
[[File:RFP,_YFP,_GFP,_CFP,_cut_E+X_10.07.12_exp.png|thumb|center|500px|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.]]
[[File:RFP,_YFP,_GFP,_CFP,_cut_E+X_10.07.12_exp.png|thumb|center|500px|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.]]
Line 150: Line 1,230:
-
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.
+
The constructs dld+RBS (<partinfo>BBa_K284003</partinfo> and <partinfo>BBa_B0034</partinfo>), Jen1+RBS (<partinfo>BBa_K284002</partinfo> and <partinfo>BBa_B0034</partinfo>), VGB+RBS (<partinfo>BBa_B0034</partinfo> and <partinfo>BBa_B0034</partinfo>) and VHB+RBS (<partinfo>BBa_K258005</partinfo> and <partinfo>BBa_B0034</partinfo>) 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.
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
{| class="wikitable" style="text-align:center;margin: 1em auto 1em auto;"
Line 652: Line 1,732:
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!
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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</div>
</div>
 +
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 +
{{:Team:NTNU_Trondheim/Templates/Sponsors}}
 +
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 +
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{{:Team:NTNU_Trondheim/Templates/Footer}}

Latest revision as of 17:15, 25 September 2012

Search and Destroy
Search and Destroy
NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Notebook Unfiltered notes from the lab


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Click on a month name or date to view the respective notebook entries. For information on plasmids and DNA mentioned in the notebook, see Stocks and Constructs. For explanations of abbreviations used, click here.


Retrieved from "http://2012.igem.org/Team:NTNU_Trondheim/Notebook"