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Lactate Sensor
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Useful sites
Site Directed mutagenisis
Tm calculator for mutagenic primers http://depts.washington.edu/bakerpg/primertemp/primertemp.html
Codon usage bias table http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-usage.html
Quick Change Primer Design Program https://www.genomics.agilent.com/collectionsubpage.aspx?pagetype=tool&subpagetype=toolqcpd&pageid=15
Tool to translate nucleotide sequence http://web.expasy.org/translate/
Other
PCR protocol http://openwetware.org/wiki/PCR
February 23, 2012
PCR amplification of the mglB gene (Glucose Binding Protein)
Reagents
- 35 uL ddH20
- 10 uL 5X phusion buffer
- 1.5 uL 10 mM dNTP's
- 1.0 uL BI 139 GBP forward primer
- 1.0 uL BI 140 GBP reverse primer
- 1.0 uL diluted template PJG 181
- 0.5 uL Phusion Polymerase
Procedure
Our forward primer was designed to include a Pst1 restriction site. Our reverse primer included an Xho1 restriction site.
Program-Phusion
1. 98 °C, 2 min
2. 98 °C, 30 sec
3. 60 °C, 30 sec
4. 72 °C, 2 min
5. repeat (2-4) 35x
6. 72 °C, 5 min
7. 4 °C, forever
March 2, 2012
PCR purification of Glucose Binding Protein (GBP)
Reagents
- 35 uL ddH20
- 10 uL 5X phusion buffer
- 1.5 uL 10 mM dNTP's
- 1.0 uL BI 139 GBP forward primer
- 1.0 uL BI 140 GBP reverse primer
- 1.0 uL diluted template PJG 181
- 0.5 uL Phusion Polymerase
Procedure
March 8, 2012
Restriction digest of GBP and PBAD
PCR Product
- 14 μL H2O
- 5 μL 10x NEB buffer 3
- .5 μL 100x BSA
- 30 μL DNA
- Mix well before adding restriction enzymes
- 1-2 μL of each restriction enzyme
- incubate at 37 °C for 1.5 hours. (overnight if needed)
March 9, 2012
Low-melt gel
Ran our restriction digests of our GBP gene as well as our plasmid to get rid of cut off DNA and any other wanted items. We did have bright DNA bands on the gel, and were successful in cutting out our DNA and getting them into labeled tubes.
March 13, 2012
Ligation of GBP and pBAD plasmid
Reagents
- 6.5 uL ddH20
- 1.5 uL 10X ligase buffer (includes ATP)
- 1.0 uL T4 DNA ligase
- 3.0 uL vector
- 3.0 uL insert
Procedure
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add 1 μL ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. - Add appropriate amount of insert to the tube.
- Add appropriate amount of vector to the tube.
- Add 0.5 μL ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting. - Let the 10 μL solution sit at 22.5°C for 30 mins
- Denature the ligase at 65°C for 10min
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20°C
March 15, 2012
Transformation of ligation into DH5α cells
16-4JR GBP transformation and control
Procedure
- Thaw DH5α cells on ice
- Place 2μL of Ligation mix into ~25μL of DH5α cells
- Vortex, or mix with flicking, then place back on ice for 5-10 mins
- Heat shock at 42 °C for 60s
- Place immediately back on ice for 2-5 mins
- Add .5mL of LB and incubate at 37 °C. (30 mins if ampicillin resistant. 60 mins everything else.)
- Plate
March 16, 2012
Streak Colonies
17-1JR
Procedure
- pick up colony with pipette tip
- place in 50 μL ddH2O0
- Streak on LB amp plate
- place streaked plate in 30 °C incubator over weekend
Arabinose Primers
17-2JR
- Forward: BI151
- Reverse: BI152
- araF gene
Colony PCR
17-3JR
- 2 μL of colony in ddH2O to --- μL master mix
- Taq polymerase
- run at Taq settings
April 27, 2012
PCR Purification
4/27JR 1
- 5:1 Buffer PB to PCR
- 200 μL Buffer to 40 μL PCR
- Centrifuge 30-60s at 13,000 rpm
- discard overflow
- add .75ml buffer PE
- centrifuge 30-60s @13,000 rpm
- discard and centrifuge again for 1 min
- place in 1.5ml tube
- add 50 μL EB buffer
- let sit for 1 min and then centrifuge 1 min
Restriction Digest
4/27JR 2
- Same protocol as March 8, 2012 for GBP
- Buffer 3. Restriction sites are pstI and XhoI
Josh's Primers
- design site directed mutagenesis primers
April 30, 2012
Low Melt Gel AraF
- 1% low melt agarose gel
- 6 μL Loading Dye
- 6 μL 1 kb ladder
- 50 μL Restriction Digest of AraF
- Run @ 90V for 52 mins.
May 1, 2012
Ligation
- control (w/out vector)
- ligation of araF low melt Restriction Digest
Transformation
- transformed plasmid into DH5α cells
- plated 500 μL each of control and araF pBAD plasmid
- placed in 30 °C incubator till Friday
May 8, 2012
Cell growth
- placed colonies 1, 3, and 4 in separate 5 mL of LB/AMP.
- Grew overnight in 37 °C incubator
May 9, 2012
Plasmid Isolation
- used plasmid mini prep kit
- isolated plasmid from 3 colonies
==May 11, 2012
Repeat cell growth
- ran nano drop on 3 cell colonies
- only had 9 ng/μL
- began re-growth of colonies 1, 3 and 4.
- placed in 5 mL LB/AMP and put in 37 °C incubator
May 12, 2012
Plasmid prep
- Took LB cells and isolated plasmid
- used plasmid mini prep kit
- obtained around 80 ng/μL
- error for each sample on N.D. machine. Will re-run Monday
May 14, 2012
Nano Drop re-read
- re-took N.D. readings with success
- 1 153 ng/μL
- 3 96 ng/μL
- 4 114 ng/μL
May 15, 2012
Sequence Prep
- placed 2 μL of each isolated plasmid
- 1 μL of forward primer for pBAD plasmid
- labeled JR 1, 3, 4. On the side ABP
May 18, 2012
Primers
- designed primers for trg/envz protein
May 21, 2012
- presentation
May 22, 2012
Colony PCR
- re-performed colony pcr with araf gene plasmids.
- ran pcr with IG 57 and IG () primers
- plated 8 colonies on ampacillin plate
May 29, 2012
Analysis Colony PCR
- 1-7 incorporated araF gene
- lane 8 did not incorporate
Growth of cells
- chose cells 1-3 and 7 to grow for isolation of plasmid which incorporated my gene
May 30, 2012
plasmid isolation
- performed plasmid mini prep w/kit
- ran nano drop and obtained 140-160 ng/μL plasmid
June 4, 2012
Sequence
- prepared 4 samples from isolated plasmids for sequencing
- 2 μL Dna
- 1 μL IG57 primer
June 5, 2012
Mutagenesis
- prepared multi-mutagenesis on plasmid 1 and 2
- ran both forward and reverse reactions for pcr
June 8, 2012
DpnI restriction
- added 1μL of DpnI restriction enzyme to each forward and reverse pcr reaction.
- incubate 6 hrs at 37 °C
June 11, 2012
Ligation
- thaw DH5α cells on ice
- add 2μL of DpnI restriction pcr reaction to cells
- vortex or flick and sit on ice 5-10 mins
- heat shock 42°C for 60 seconds
- place immediately on ice 2-5 mins
- add .5mL of LB
- incubate 37°C for 30 mins
- plate
June 18, 2012
Incubation
- Had 3 colonies on ABP mutation Reverse #1
- incubate colony in 5 mL overnight
June 19, 2012
Plasmid prep and sequence
- used plasmid kit to isolate plasmids with mutations
- placed 2 μL of DNA w/ 1 μL primers IG57 and IG12
Lactate Sensors
September
September 4,2012
We took over from Josh and Jeffrey’s work and figured out their primer design and sequences (see below). Since they had already used the Strategene Multi- site directed mutagenesis kit to introduce the first 3 primers, but then sequenced or 4 colonies but could only find clones with 2 of the 3 mutations, we decided to just try sequencing more colonies on the plate and started overnights for plasmid purification (8 more colonies).
>MglB
atgAATAAGA CGGTGCCGCT AGTACGACGA TCTGTAGTGC TCAGCTGCTG ATGAATGATT ATTGCTGGCG AAAGGGGTGA TACGGTGATT GAGAAAGCGC GTCTCGTAAG GCGCTGGATA GTCCGGCATT ATTCAAGGCG TCTGAACAAA GACGGTCAGA TGCAGAAGCA CGTACCACTT ACAGTTACAG TTAGATACCG CTGGCTGTCT GGCCCGAACG GGCAATGGGC GCGGTTGAAG CGTCGATGCG CTGCCAGAAG ACTGAACGAT GCTAACAACC TGGTAAAGGT GCGGCTGATG TTATGTTGGC GTAGATAAAG
AGGTGTTAAC CCTGTCTGCT GTGATGGCCA GCATGTTATT GCACACGCTG CTGATACTCG CATTGGTGTA ACAATCTATA TAACTTTATG GCAAGGCTAT TGAGCAAGAT GCGAAAGCCG CGCCAGATGT
CTCAGAATGA CCAGTCCAAG CAGAACGATC AGATCGACGT
AGGCACTGGC AATCAACCTG GTTGACCCGG CAGCTGCGGG
GTGGGCAAAA CGTGCCGGTG GTTTTCTTCA ACAAAGAACC
GCTACGACAA AGCCTACTAC GTTGGCACTG ACTCCAAAGA
ATTTGATTGC TAAACACTGG GCGGCGAATC AGGGTTGGGA
TTCAGTTCGT ACTGCTGAAA GGTGAACCGG GCCATCCGGA
ACGTGATTAA AGAATTGAAC GATAAAGGCA TCAAAACTGA
CAATGTGGGA CACCGCTCAG GCGAAAGATA AGATGGACGC
CCAACAAAAT CGAAGTGGTT ATCGCCAACA ACGATGCGAT
CGCTGAAAGC ACACAACAAG TCCAGCATTC CGGTGTTTGG
CGCTGGCGCT GGTGAAATCC GGTGCACTGG CGGGCACCGT
AGGCGAAAGC GACCTTTGAT CTGGCGAAAA ACCTGGCCGA
GCACCAACTG GAAAATCGAC AACAAAGTGG TCCGCGTACC
ACAACCTGGC TGAATTCAGC AAGAAAtaa
- mutant MglB Y10K, D14K, N91K, K92L, H152M, D154H, R158K, W183K, D236A ,
N256D
BI157 Y10K, D14K CGC ATT GGT GTA ACA ATC AAA AAG TAC GAC AAA AAC TTT ATG TCT GTA GTG Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 10 from Y to K and number 14 from D to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI158 CAC TAC AGA CAT AAA GTT TTT GTC GTA CTT TTT GAT TGT TAC ACC AAT GCG Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 10 from Y to K and number 14 from D to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI159 N91K, K92L GTG CCG GTG GTT TTC TTC AAA CTG GAA CCG TCT CGT AAG Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 91 from N to K and number 92 from K to L. One of 12 primers designed to mutate GBP to GBP.G1.
BI160 CTT ACG AGA CGG TTC CAG TTT GAA GAA AAC CAC CGG CAC Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 91 from N to K and number 92 from K to L. One of 12 primers designed to mutate GBP to GBP.G1.
BI161 H152M, D154H, R158K G AAA GGT GAA CCG GGC ATG CCG CAT GCA GAA GCA AAA ACC ACT TAC GTG Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 152 from H to M, number 154 from D to H, and number 158 from R to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI162 CAC GTA AGT GGT TTT TGC TTC TGC ATG CGG CAT GCC CGG TTC ACC TTT C Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 152 from H to M, number 154 from D to H, and number 158 from R to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI163 W183K CAG TTA GAT ACC GCA ATG AAA GAC ACC GCT CAG GCG Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 183 from W to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI164
CGC CTG AGC GGT GTC TTT CAT TGC GGT ATC TAA CTG Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 183 from W to K. One of 12 primers designed to mutate GBP to GBP.G1.
BI165 D236A GTG TTT GGC GTC GCG GCG CTG CCA GAA G Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 236 from D to A. One of 12 primers designed to mutate GBP to GBP.G1.
BI166 CTT CTG GCA GCG CCG CGA CGC CAA ACA C Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 236 from D to A. One of 12 primers designed to mutate GBP to GBP.G1.
BI167 N256D GCG GGC ACC GTA CTG GAT GAT GCT AAC AAC CAG Forward mutagenic primer for mglB gene in E. coli. Changes amino acid number 256 from N to D. One of 12 primers designed to mutate GBP to GBP.G1.
BI168 CTG GTT GTT AGC ATC ATC CAG TAC GGT GCC CGC Reverse mutagenic primer for mglB gene in E. coli. Changes amino acid number 256 from N to D. One of 12 primers designed to mutate GBP to GBP.G1.
IG151 araf E. coli. ccgctgcagaggaggtatatattatgcacaaatttactaaagccc This is the forward primer for the E. Coli periplasmic arabinose binding protein. It includes the PstI restriction site, shine delgarno and first 22 bases. This will be used to be mutated to bind lactate instead of arabinose. Reverse primer is IG152.
IG152 araf E. coli. cggctcgagttacttaccgcctaaaccttt This is the reverse primer for E. coli periplasmic arabinose binding protein. This includes the XhoI restriction site as well as the last 21 bases. This will be used to be mutated into a lactate sensor. The forward primer is IG151.
Primers to mutate AraF to a lactate sensor to incorporate the following mutations: (W16Y, C64A, D89L, D90A, M108A, L145R, Y147S, R151S, M204Y, N232K )
>AraF sequence
atgCACAAAT GTCACAATCC GCTATGGCGG GCCGTGGTTC CAGACCGAAT GGTTATTAAG ATTGCCGTGC TGCCAGTGGC GCAAAAGGTT CGTCGCGAAA GCGCGTGGCT CGCCAAAGGT AAGCCAATGG CGAACGTCAG GGCCAGGAAC AAGCGCGGTG ATGGCGATTA ATCTATGGAT GCGCTGAAAG CAAATCTAAC GACATCCCGG GGAAGTTAAA CATTGGCTGA CGCGACGGAA GGTCAGGGCT GGATGCGGTG AGCGAACTGT AAGCCCGGAC GTACATGGCT CGTTGAACCG CCAAAATTTA TAAAGAAGAA CTGGAGAAAA
BI179 (W16Y) CaaccggaagagccgTATttccagaccgaatgg This is for site directed mutagenesis of the araf gene to convert arabinose binding protein to bind lactate. Converts A.A. 16 from tryptophan to tyrosine.
BI180 (C64A) gcaaaaggtttcgttattGCGactccggacccgaaactc This is for the site directed mutagenesis of the araF gene. This changed the binding of arabinose to the binding of lactate. Changes A.A. 64 from cysteine to alanine.
TTACTAAAGC CCTGGCAGCC ATTGGTCTGG CAGCCGTTAT
AGAACCTGAA GCTCGGTTTT CTGGTGAAGC AACCGGAAGA
GGAAGTTTGC CGATAAAGCC GGGAAGGATT TAGGGTTTGA
CGGATGGCGA AAAAACATTG AACGCGATCG ACAGCCTGGC
TCGTTATTTG TACTCCGGAC CCCAAACTCG GCTCTGCCAT
ACGATATGAA AGTCATTGCC GTGGATGACC AGTTTGTTAA
ATACCGTTCC GCTGGTGATG ATGGCGGCGA CTAAAATTGG
TGTATAAAGA GATGCAGAAA CGTGGCTGGG ATGTCAAAGA
CCGCCAACGA ACTGGATACC GCCCGCCGCC GTACTACGGG
CGGCCGGATT CCCGGAAAAA CAAATTTATC AGGTACCTAC
GGGCATTTGA CGCTGCCAAC TCAATGCTGG TTCAACATCC
TCGTCGGTAT GAACGACAGC ACCGTGCTGG GCGGCGTACG
TTAAAGCGGC CGATATCATC GGCATTGGCA TTAACGGTGT
CTAAAGCACA GGCAACCGGC TTCTACGGTT CCCTGCTGCC
ATAAATCCAG CGAAATGCTT TACAACTGGG TAGCAAAAGA
CCGAAGTTAC CGACGTGGTA CTGATCACGC GTGACAACTT
AAGGTTTAGG CGGTAAGtaa
BI181 (D89L, D90A) gatatgaaagtcattgccgtgCTGGCGcagtttgttaacgccaaaggt This is for the site directed mutagenesis of the araf gene. Converts the binding of arabinose to the binding of lactate. Changes A.A.s 89-90 from aspartic acid to leucine and alanine.
BI182 (M108A) gttcctctggtgatgGCGgcggcgaccaaaatt This is for the site directed mutagenesis of the araf gene of E. coli. This converts the binding of arabinose to the binding of lactate. Changes A.A. 108 from methionine to alanine.
BI183 (L145R, Y147S, R151S) attacctccaacgaaCGTgatTCTgctcgtcgcTCTactacgggttctatg This is for the site directed mutagenesis of araf gene. Changes binding of arabinose to lactate. Changes A.A.s 145, 147, and 151 from Leucine, tryosine and arginine to arginine and serine twice. Forward primer
BI184 (M204Y) tggctgatcgtcggtACCaacgacagcaccgtg Forward primer for site directed mutagenesis of araf gene. Converts binding of arabinose to binding of lactate. Changes A.A. 204 from methionine to tyrosine.
BI185 (N232K) atcggtatcggtattAAAggtgtggatgcggtg Forward primer for site directed mutagenesis of araf gene. Converts binding of arabinose to binding of lactate. Changes A.A. 232 from asparagine to lysine.
BI186 CCA TTC GGT CTG GAA ATA CGG CTC TTC CGG TTG Reverse primer for site directed mutagenesis of araf. Complement to forward primer BI179.
BI187 GAG TTT CGG GTC CGG AGT CGC AAT AAC GAA ACC TTT TGC Reverse primer for the site directed mutagenesis of araf gene. Reverse complement to primer BI180
BI188 ACC TTT GGC GTT AAC AAA CTG CGC CAG CAC GGC AAT GAC TTT CAT ATC Reverse primer for site directed mutagenesis of araf gene. Reverse complement to primer BI181 BI189 AAT TTT GGT CGC CGC CGC CAT CAC CAG AGG AAC Reverse primer for site directed mutagenesis of araf gene. Reverse complement to primer BI182
BI190 CAT AGA ACC CGT AGT AGA GCG ACG AGC AGA ATC ACG TTC GTT GGA GGT AAT Reverse primer for site directed mutagenesis of araf gene. Reverse complement to primer BI183 BI191 CAC GGT GCT GTC GTT GGT ACC GAC GAT CAG CCA Reverse primer for site directed mutagenesis of araf gene. Reverse complement to primer BI184 BI192 CAC CGC ATC CAC ACC TTT AAT ACC GAT ACC GAT Reverse primer for site directed mutagenesis of araf gene. Reverse complement to the primer BI185.
Sept. 5, 2012
Plasmids of MglB and AraF were submitted for sequencing
Sept. 10, 2012
We received the sequencing back form MglB and AraF and aligned sequences using seqeuncher. Of the 8 sequenced plamsids , 3 for MglB had incorporated the mutations from all 3 primer (clone 2, 3 and 7). For AraF, only clone AF1 had incorporated all mutations from all 3 primers. These plasmid were then used to set up a new round of mutagenesis using the Strategene Multi-site directed mutagenesis kit and the rest of the primers. Two reactions were setup for MglB (MglB clone 7 was used as template) and two for AraF (AraF clone 1 was used as template). One reaction consisted of the remaining forward primers, while the other used the reverse primers.
Sept. 11, 2012
PCR reactions were digest with DpnI according to the Strategene protocol
Sept 12, 2012
PCR reactions were transformed and selected on LB-AMP according to the Strategene protocol
Sept. 13, 2012
8 Colonies from each reaction (for a total of 36 colonies) were started as overnights in LB-AMP
Sept 14, 2012
Plasmid preps were performed on each of the 32 overnights. Plasmids were then submitted for sequencing of the MglB2 and AraF genes.
Sept. 17, 2012
Sequencing results are back. Of the 16 total plasmids for both MglB2 and AraF, only 3 plasmids of Mgl2 incorporated the mutations from all of the primers (without any additional mutations), while only 1 of the AraF did! Hey- at least we got it
Sept 18, 2012
The Mgl2 and AraF genes now have to be moved into a Cam R plasmid with a p15 origin of replication (pACYC184), so that we can introduce both the Mgl2 and AraF mutants into E.coli along with the OmpC-lacZ reporter and TrZ plasmid (AmpR) that we received from Dr Hazelbauer’s lab at the university of Missouri. Phusion PCR amplify inserts with pBAD forward primer (BI35 and MglB or AraF reverse). Use the Mgl2 and AraF mutant plasmids (BI43 and 44 respectively) as template.
Sept 19, 2012
PCr is run on gel and we get ~2 kb product as expected. PCR purify then setup restriction digests of pACYC184 and inserts.
Sept 20, 2012
Gel purify and then ligate and transform.
Sept 21
Clony PCR to check for inserts. Four of 8 colonies have the correct insert. Start overnights in LB-AMP
Sept 22
Purify plasmid and transform into E.coli long with the Trz-OmpC-lacZ plasmid from Dr Hazelbauer’s lab. Select on LB-AMP-CAM
Sept. 24
Start overnights of strain in LB-AMP-Cam and LB-AMP-Cam-Arabinose and LB- AMP-Cam-Arabinose-lacate (100mM)
Sept. 25
Perform B-gals using standard protocol
