Team:NRP-UEA-Norwich/Week9

From 2012.igem.org

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. Alpha cells that we had previously plated was inoculated into 5ml LB media ready for setting up a calibration curve for the growth study. These were grown at 37 degrees.
. Alpha cells that we had previously plated was inoculated into 5ml LB media ready for setting up a calibration curve for the growth study. These were grown at 37 degrees.
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'''working mathmatical modelling'''
 
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[[File:Comparator_circuit_equasion_iGEM_1_12.09.15.jpg | 200px | right]]
 
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E = the expression of one of the fluorescent protiens (RFP) when there is transcription of the CFP RNA at any particular level as a proportion of the expression of RFP at the same transcription rate when none of the CFP RNA  is present within the cell.  So if the promoter (PYEAR) attached to the rfp and construct 1  was expressing at a constant rate with promoter 2 entirely switched off then promoter 2 started transcribing and the amount of rfp in the cell halved then the value of EA would be 0.5  at that transcription rate of CFP (0-1)
 
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L = the length of the DNA strand that is transcribed (Leader and protein coding region) .
 
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C = the rate of transcription
 
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L/C = the period of time taken for transcription to take place, the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another
 
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A = the rate of transcription of promoter 1 (the PYEAR) as a proportion of it’s maximum possible transcription rate (0-1) 
 
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A*(L/C) = the number of RFP RNAs that can be translated independently of the presence of other RNA at any one time and so is proportional to translation (and expression) from DNA ascociated RNA (RNA that is still being transcribed).  This occurs both when the CFP RNA is and isn’t present so has to be on both the top and bottom of the equation.
 
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H = Half life of the RNA after transcription
 
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L/C + H = the full time for which the RNA would be translated assuming no interactions between leaders for instance when only one of the promoters is inducing transcription.   
 
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B = the transcription of the second promoter within the cell as a proportion of the maximum possible transcription of that promoter (0-1)
 
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Because there can not be negative expression of A (only positive expression of B to represent negative expression of A) the translation of the RFP RNA(A) = A - AB .
 
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It also has to be remembered that there will never be full interaction between the two RNA leaders, particularly at low concentrations if only because the two strands never come in to proximity or because of cellular processes; consequently  the function B/(D+B) must be used giving the formula; translation of the RFP RNA(A) = A – A (B/(D+B))
 
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D is a constant of the biological system whose derivation is so complex that it can only really be calculated through observation but can be modelled at various levels.
 
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So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal EA ; this indicates the degree of attenuation of one RNA from the other.
 
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To get the degree of translation of the other RNA (EB) just swap A for B throughout the equation.
 
==Day 3==
==Day 3==

Revision as of 11:51, 15 September 2012

Header1NewGreen.png

NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

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

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

Contents

Week 9

Day 1

. We looked at the plates of transformant colonies of RFP and GFP that have kanamycin resistance. Both Rebecca's and Rachel's plates for GFP looked a little dodgy. Rebecca's had not grown at all whereas, Rachel's had but the colonies were rather large. The RFP colonies looked good. However, after double checking in the parts registry, we realised that these BioBricks have no RBS and therefore, we decided to keep these plates for now and went back to the previous BioBricks used with RFP, GFP and CFP.

. Russell, Rebecca and Khadija used Pst1 and Xba1 to do a double restriction digest of RFP, GFP and CFP that we previously used. We decided that despite the backbone fragment seemingly being too large, we would try it out. Into each eppendorf a 3.2µl mastermix sample of 5µl Pst1, 5µl Xba1, 2µl of BSA ad 20µl Buffer H. There were 9 eppendorf tubes containing: 3.6µl of DNA and 13.2µl of water (CFP1), 6.03µl of DNA and 10.77µl of water (CFP2), 3.47µl of DNA and 13.32µl of water (RFP1), 3.92µl of DNA and 12.88µl of water (RFP2), 3µl of DNA and 13.8µl of water (GFP1), 3.91µl of DNA and 12.89µl of water (GFP3) and 3.74µl of DNA and 13.06µl of water (GFP4). These were left overnight.

. At the same time, as we were using isolated plasmid DNA we decided to transform the original DNA into alpha competent silver standard cells from Bioline.

. The synthesised DNA for Constructs 1 and 2 that Khadija and Pascoe had designed a few weeks ago, for the comparator circuit finally arrived!. Immediately, we got to work on it, Rebecca, rehydrated the DNA and transformed it into Bioline alpha select gold standard competent cells. Plates were made and incubated overnight at 37 degrees.

Day 2

. The plates from the transformation of potentially our next two BioBricks, Construct 1 and 2 were looked at. The plates showed good growth. The negative control showed no growth as expected. The positive control of M-B that was still in pUC57 transformed cells showed good growth. The construct plates showed very good growth and immediately Russell innocuated into LB media containing ampicillin. This was grown for a few hours and we found these were sufficiently cloudy so Rebecca and Khadija reinoculated 10µl of these colonies (4 of Construct 1 and 4 of Construct 2) into 2 new tubes each. In total we have 32 tubes of Constructs 1 and 2. These were all returned to the incubator for further incubation.

. The restricted reporter proteins: GFP, RFP and GFP were run on a 1.2% gel to isolate the fragment. We found that the RFP samples we had were the only successfully cut fragments. The others lanes containing GFP and CFP showed no signs of DNA. Khadija cut the RFP fragments out of the gel and Rachel used the Promega Wizard Kit and protocol to purify the DNA.

. Having had little success with the BioBricks of GFP, RFP and CFP after the original transformation in week 3. We decided to use the original plates to streak the DNA. We located the plates which by now have hardened somewhat. Russell streaked the colonies onto fresh ampicillin plates. These were incubated overnight at 37 degrees celsius.

. As we are running low on ready made LB agar, we made extra supplies. In total we made 7 glasses of 250ml agar which were autoclaved.

. Alpha cells that we had previously plated was inoculated into 5ml LB media ready for setting up a calibration curve for the growth study. These were grown at 37 degrees.

Day 3

. From the streaked RFP and CFP plates, Russel inoculated 6 colonies of RFP and 6 colonies of CFP into LB media containing ampiciilin resistance. These were grown overnight at 37 degrees.

. The samples we requested from Life Biosciences were delivered, unfortunately not all the samples we wanted were returned. We called up to request the rest of the samples. Further liason will occur. In the meantime, with using 1µl of BM1, Rebecca transformed 30µl of Bioline alpha select gold standard cells and incubated them at 37 degrees celsius over night.

. PyeaR + GFP transformed cells that we had previously grown on a plate was inoculated by Russell into culture and incubated overnight at 37 degrees.

. As a follow up on the growth study that we did in week 7, we made a calibration curve using LB media as a zero and measuring absorbances at 600nm. The calibration curve was set up using the alpha cells we had grown overnight. The culture was spun down alike in the growth study. The cells peletted was resuspended in LB media and made up to different concentrations. The concentrations themselves were not important. The absorbances were recorded and the from cuvettes, Rebecca plated 20µl of the culture with 180µl of LB media and grew these overnight in a 37 degree incubator.

. We identified the plate contained the M-B (in pSB1C3) transformed colonies which we originally inoculated and attained the MB1-4 that we sent off for sequencing. We inoculated the colony which later became MB2 and was proven by sequencing to be what we wanted. Along with this colony, 9 other colonies were inoculated. As MB1, 3 and 4 were not the sequenced we wanted, we scrapped these. Only MB2 that was labelled is the same colony as any previously inoculated. The others are all new colonies. Later these tubes were reinoculated into 1 other tube so we had 20 tubes. These were all incubated at 37 degrees.

. The grown overnight cultures of Constructs 1 and 2 were miniprepped by Rebecca and Khadija. These were then nanodropped. Thee following concentrations were found: (C1 = Construct 1, C2 = Construct 2. The following number refers to the isolate colony which was marked on the plate and the a and b refer to the tubes we used during mini prepping (2 5ml inoculations were miniprepped into one eppendorf- essentially a and b should contain the same DNA. We separated them due to the protocol stating using 1-10ml)

C1-1a: 523.9 ng/µl

C1-1b: 421. 2ng/µl

C1-2a: 456.5 ng/µl

C1-2b: 422.8 ng/µl

C1-3a: 571.8 ng/µl

C1-3b: 389.0 ng/µl

C1-4a: 438.0 ng/µl

C1-4b: 316.6 ng/µl


C2-1a: 448.9 ng/µl

C2-1b: 546.9 ng/µl

C2-2a: 900.0 ng/µl

C2-2b: 633.0 ng/µl

C2-3a: 572.5 ng/µl

C2-3b: 530.7 ng/µl

C2-4a: 617.7 ng/µl

C2-4b: 548.7 ng/µl


. Spurred on by the high concentrations and a new record by Rebecca, we set up a restriction digest to isolate the constructs to ligate into pSB1C3. The double restriction used EcoR1 and Pst1. A master mix was set up using: 4µl of BSA, 40µl of Buffer H, 10µl of EcoR1 and 1µl of Pst1. Into eppendorfs labelled with what Construct DNA, 3.2µl of master mix was put in. These eppendorfs had DNA and nuclease free water pippetted in, in advance. The following amounts of DNA were added and water was added to make the samples up to 20µl.

C1-1a: 1.91 µl

C1-1b: 2.37 µl

C1-2a: 2.19 µl

C1-2b: 2.37 µl

C1-3a: 1.75 µl

C1-3b: 2.57 µl

C1-4a: 2.28 µl

C1-4b: 3.16 µl


C2-1a: 2.23 µl

C2-1b: 1.83 µl

C2-2a: 1.11 µl

C2-2b: 1.58 µl

C2-3a: 1.75 µl

C2-3b: 1.88 µl

C2-4a: 1.62 µl

C2-4b: 1.82 µl

The restriction digest was left in a 37 degree water bath overnight.

. Additional Agar and LB media was made and autoclaved. The LB media was pipetted into 5 ml bottles before autoclaving.


Day 4

. Looking at the plates that we transformed yesterday, we saw that the plates were good. The 20µl and 200µl plates of BM1 transformed E.coli contained many colonies. These colonies were compared to those on the control plates. The negative control of just cells showed no colony growth. The positive control of PyeaR + GFP transformed cells, which also contain chloramphenicol resistance showed similar growth to that of the BM1's. From these plate 4 colonies were inoculated into LB media containing chloramphenicol resistance.

. The overnight inoculations of M-B were miniprepped. Before miniprepping, 10µl of the originally inoculated samples were reinoculated into new media to maintain an ongoing culture. as we were rushing to have MB2 sent for sequencing, we miniprepped MB2 and had in nanodropped first. The concentration was found to be 58.2ng/µl.

. The restricted DNA of Constructs 1 and 2 were run on a gel to isolate the actual fragment. Due to insufficient amounts of agarose, only one gel was made and as we have 16 samples and a ladder, only the 'a' samples were run (e.g C1-1a, C2,4a, etc). The remaining was put in the fridge. The gel was made to be denser as the fragment to be isolated is only 171bp. The gel was made to be 1.5% w/v. It was run for an hour. It was found that the the ladder had not gone into the lanes. For each sample of DNA, 5µl of loading dye was added to 20µl of DNA. As we had got a new ladder (100bp ladder) we were unaware that loading dye was not already added. Therefore, there was no ladder, we were also perplexed by the presence of 3 bands, though the lowest we suspect to be shadowing. Also two samples of Construct 1 did not successfully digest (C1-2a and C1-3a). The rest were good. We cut the DNA fragments from the gel and stored them in the fridge overnight.

. Russell reinoculated lots and lots of RFP and CFP. From the original tubes inoculated 2 others were inoculated, totaling 3 tubes for each colony (total of 6 colonies in tubes for each of RFP and CFP)


Day 5

Dry Lab

. Joy researched the amount of DNA we would need to send for sequencing and calculated how much DNA would be need to be sent off to match the requirements.

Labs

. The miniprepped M-B was nano dropped and the following concentrations were found:

MB1: 78 ng/µl

MB3: 169.3 ng/µl

MB4: 91.7 ng/µl

MB5: 113.0 ng/µl

MB6: 68.2 ng/µl

MB7: 79.2 ng/µl

MB8: 32.2 ng/µl

MB9: 132.2 ng/µl

MB10: 147.6 ng/µl

MB6 and MB 8 had abnormally high 260/2800 readings at 4.19 and 22.85 respectively.

. With more agarose, we made another 1.5% w/v gel and the rest of the Construct 1 and 2 samples were run on a gel for an hour. This time, loading dye was added to the ladder. For each sample of DNA, 5µl of loading dye was added to 20µl of DNA/ladder. This time the ladder ran, again, we noticed the shadowing. Counting the number of bands and their relative distances we could be sure that the last band was shadowing as the shortest marker fragment was also shadowed. The fragments were the right size and these were cut out before being purified.

. The gel slices of Construct 1 and 2 fragments were purified using Promega Clean Up kit. After purification, these were nanodropped. The concentrations were found to be as follows:

C1-1a: 15.1 ng/µl

C1-1b: 4.5 ng/µl

C1-2b: - 9.0 ng/µl

C1-3b: - 7.2 ng/µl

C1-4a: 14.0 ng/µl

C1-4b: - 0.4 ng/µl


C2-1a: 7.6 ng/µl

C2-1b: 1.1 ng/µl

C2-2a: 6.3 ng/µl

C2-2b: 0.4 ng/µl

C2-3a: 14.6 ng/µl

C2-3b: 2.2 ng/µl

C2-4a: 14.0 ng/µl

C2-4b: -3.2 ng/µl

All samples containing less than 4ng/µl of DNA were discarded.


. Following the low results of Construct 1 and 2, Rebecca repeated the double restriction digest, however, as there was a slight communication error, the wrong enzymes were added. Instead of Pst1 and EcoR1, Pst1 and Xba, was added instead. After 5 hours of digestion, we tookk these out and refrigerated them, in case they would be needed for later cloning. Also after some discussion with the advisors, we decided the the concentrations ascertained should be sufficient to carry out the ligation.

. The PyeaR samples that were inoculate on Wednesday were plated and incubated at 37 degrees overnight and taken out the next day. This is in preparation for next week when we hope to characterise the PyeaR + GFP BioBrick further by ascertaining some results of growth in potassium nitrate through the use of a fluorimeter.

. Rachel miniprepped the BM1 that had been inoculated by Russell on Thursday. Before miniprepping, 5µl was removed and reinoculated. The next day these reinoculations would be taken out of the incubator and placed in the fridge. The miniprepped DNA using the Promega DNA isolation kit was nanodropped and found that there was very little DNA. The was 13.1ng/µl. Again after discussion with advisors, we feel that this may be sufficient and a ligation will be carried out on Monday.

. Khadija miniprepped all the RFP and CFP's. In totally 3 bottles of each colony was spun down into a pellet and then the rest followed the protocol of the Promega kit. Following the miniprep all these samples of isolated DNA was nanodropped to identify the concentration. The following concentrations were found:

RFP1 (1): 113.9 ng/µl

RFP1 (2): 123.9 ng/µl

RFP1 (3): 126.1 ng/µl

RFP2 (1): 67.1 ng/µl

RFP2 (2): 201.8 ng/µl

RFP2 (3): 107.7 ng/µl


CFP1 (1): 239.3 ng/µl

CFP1 (2): 246.4 ng/µl

CFP1 (3): 170.9 ng/µl

CFP2 (1): 48.4 ng/µl

CFP2 (2): 218.0 ng/µl

CFP2 (3): 163.9 ng/µl