Team:SYSU-China/Notebook/Labnotes
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Promoter EF1a we used to drive regulationg parts of tet-on or tet-off system are 537bp in length(supported by professor Lei xiao, Zhejiang University).By NCBI blast search,first 349bp of the 537bp-pEF1a was found to be CPPT,one of the essential elements of inserting backbone plasmid of lentivirus. While the function of CPPT cassette inside pEF1a is yet to figure out, the result of molecular construction suggested that CPPT, which exists both inside 537bp-pEF1a and p199 backbone plasmid, has high ratio of recombination that can interfere the normal inserting process of lentivirus vector and the expression of downstream genes. | Promoter EF1a we used to drive regulationg parts of tet-on or tet-off system are 537bp in length(supported by professor Lei xiao, Zhejiang University).By NCBI blast search,first 349bp of the 537bp-pEF1a was found to be CPPT,one of the essential elements of inserting backbone plasmid of lentivirus. While the function of CPPT cassette inside pEF1a is yet to figure out, the result of molecular construction suggested that CPPT, which exists both inside 537bp-pEF1a and p199 backbone plasmid, has high ratio of recombination that can interfere the normal inserting process of lentivirus vector and the expression of downstream genes. | ||
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<br />Figure 10. The recombination zone<br /><p> | <br />Figure 10. The recombination zone<br /><p> | ||
High-ratio recombination between the 2 copies CPPT in plasmid p199-pEF1a(537bp)-gene was first suspected as the forward sequencing primer often present 3 kinds of sequencing results: | High-ratio recombination between the 2 copies CPPT in plasmid p199-pEF1a(537bp)-gene was first suspected as the forward sequencing primer often present 3 kinds of sequencing results: |
Revision as of 09:06, 27 September 2013
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Molecular construction of the up stream elements of tet systems and suicide genes.
Introduction
Team members in Charge:Dawei He,Yiming Fang and Haoqi Chen
Author: Dawei He
Despite a good comprehension of the knowledge of Molecular biology and its relating experimental technologies,We never came to and stay in a Molecular biology lab and were completely lack of any experience. To achieve the designated tasks in extremely insufficient time(less than 3 months),We had to learn fast and start our work independently as soon as possible.
2013.5.20-5.23
Contents: Contact and learning of Molecular construction
Team members in Charge:Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)
1. DNA extraction techniques: Plasmid extraction, DNA clean up(extraction from PCR or restriction endonuclease digestion systems,extraction from Ag gel, etc)
2. PCR techniques:
Primer(for molecular construction or sequencing) designing, PCR for molecular construction, colony PCR for verification.
3. Endonuclease digestion
2013.5.24 to 5.31
Contents: First plasmid construction:pCDNA3.0-PGK-BSD
Annotation:pCDNA3.0 is a backbone plasmid ideal for transient expression in our designated cell lines including HEK293T,HepG2 and Bocs. All elements of our project are first to be incised into pCDNA3.0 and tested during transient expression in cell lines. Driven by promoter PGK,BSD functions as a resistantgene specially for our up-stream tet-system during stable transfection by lentivirus)
Figure 1.Backbone plasmid pCDNA3.0(completed featured) align="center"
Figure 2.pCDNA3.0-PGK-BSD
construction details:
1.PCR of PGK-BSD(915bp in length) from donor plasmid(as PCR template)
Primers:Forward primer with restriction site BamHI and necessary base pairs from 5’ end
Reverse primer with restriction site XhoI and necessary base pairs from 5’ end
Bases pairs should appear at both flanks of restriction sites to provide anchoring sites for endonuclease,3’ end of the restriction sites in a primer is already covered by base pairs, so some base pairs must be added to its 5’ end. Without such manually added base pairs from 5’ end, efficiency of endonuclease digestion of PCR product will be significantly reduced.
1.1 PCR system set up
5×fastpfu PCR buffer | 5μl |
2.5mM dNTP: | 2.5μl |
10uM PB F primer | 0.8μl |
10uM PB R primer | 0.8μl |
Template(plasmid) | 4μl(5.36ng/ul,21.4ng in total. Acceptable quantity from 5-30ng) |
Fastpfu DNA polymerase | 0.5μl |
ddH20 | 11.4μl(up to 25ul) |
1.2 temperature program set up
Annealing temperature in main PCR cycles =Primer Tm-5℃
Pre-cycles with lower annealing temperature in PCR process of primers with restriction sites(or any primers that include bases pairs not complementary to their original templates ) are necessary to achieve both specificity and quantity demand of PCR product.
During the first PCR cycle, primer binds only and incompletely to its original templates, so tm is lower compared to the one when primer binds total- complementarily to its product generated since the first cycle, which was why the idea of adding pre-cycles before main cycles came up. Although annealing temperature in pre-cycles can be calculated to details, the temperature 15℃ below annealing temperature in main cycles will be quite universal.
Primer binding to its original template
Primer binding to its PCR product
1.3 Endonuclease digestion of backbone plasmid and insert fragment(PCR product)
PCR product PGK-BSD was purified using PCR clean up kit.
10xFermentas Buffer G | 3μL |
pCDNA3.0 | 2μL(3500ng) |
BamHI(Fer) (100% activity) | 1μL |
XhoI(Fer)(50-100% activity) | 2μL |
ddH20 | 22μL(up to 30μL) |
10xFermentas Buffer G | 3μL |
PGK-BSD | 9μL(900ng) |
BamHI(Fer) (100% activity) | 1μL |
XhoI(Fer)(50-100% activity) | 2μL |
ddH20 | 15μL(up to 30μL) |
Both digestion systems were incubated in 37℃ for 2h
Figure 3.
Result:backbone plasmid pCDNA3.0 was fully digested,but as the fragment excised from pCDNA3.0 was too short(which means too small in quality because it has the same amount of substance as the backbone’s) in length(less than 50bps),it cannot be identified in agarose gel-electrophoresis. When digested in a least one restriction site, plasmid turns from supercoil status to linearized and has lower mobility in gel-electrophoresis, so it can only be judged that backbone was definitely complete digested in at least one restriction site. Such assessment applies also to digestion of PCR product from whom the excised parts are almost never more than 50bps.
1.4 Ligation
Ligation type:1 vector+1 insert fragment
Desired ratio of the amount of substance:
Vector:insert fragment =1:5
10xFermentas T4 ligation buffer | 2μL |
BamHI-(pCDNA3.0)-XhoI | 1μL(60ng) |
BamHI-(PGK-BSD)-XhoI | 2μL(60ng) |
Fermentas T4 ligase | 1μL |
ddH20 | 12μLup to 20μL) |
Ligation for 2h at room temperature(25℃)
1.5 Transformation of ligation product
Transformation system
DNA(ligation system) | 2μL |
Competent E.coli(Top10 strain) | 50μL |
After 2h ligation,ligation system,as well as Competent E.coli taken from -80℃ storage, was first incubated in ice for 5min before mixing together.
Transformation system continued to be incubated in ice for 30mins,42℃heat shock for 60s,then again ice incubation for 5mins.
Transformation system was added 600μL antibiotic-free LB medium and put in 37℃ thermostat shaker for 30mins-resuscitation(expression of Amp resistance protein)
Transformation system was then centrifuged at 130,000g for 3mins.All but little supernatant was kept to suspend the E.coli,which was later transferred to the LB agar plate. The plate was put in 37℃ thermostat incubator overnight for colonies growth.
1.6 Verification of the colonies(each as a single clone)
Figure 4.
colony PCR set up
5×fastpfu PCR buffer | 5μl |
2.5mM dNTP | 2.5μl |
10uM PGK-BSD F primer F primer | 0.8μl |
10uM PGK-BSD R primer | 0.8μl |
Template (single-clone colony) | 1μL(taken from the single-clone Colony suspended with 5μL ddH20 |
Fastpfu DNA polymerase | 0.5μl |
ddH20 | 11.4μl(up to 25ul) |
Figure 5
Result:Clear band around 1000bp appeared for every 12 clone.
1.7 Plasmid extraction and verification
6 of the single-clone colonies were selected and added to 5ml Amp-LB medium and put to 37℃ thermostat shaker.
The bacteria were centrifuge after 16h-growth in the thermostat shaker.Plasmids were extracted using endotoxin-free kit.
The plasmids were later digested by BamHI and XhoI for verification.
10xFermentas Buffer G | 2μL |
pCDNA3.0 | 0.5μL(400ng) |
BamHI(Fer) (100% activity) | 0.3μL |
XhoI(Fer)(50-100% activity) | 0.6μL |
ddH20 | 16.6μL(up to 30μL) |
Figure 6
Result: Clear band around 1000bp appeared. Plasmids from every 4 clones were verified.
Plasmids were later sent for sequencing
2013.6.1-6.14
Construction of pCDNA3.0-pEF1a-tTA-PGK-BSD
pCDNA3.0-pEF1a-rtTA-PGK-BSD
pCDNA3.0-pEF1a-tTA advanced-PGK-BSD
pCDNA3.0-pEF1a-eGFP-PGK-BSD
Figure.7
PCR system set up
5×fastpfu PCR buffer | 5μl |
2.5mM dNTP: | 2.5μl |
10uM PB F primer | 1.6μl |
10uM PB R primer | 1.6μl |
Template(plasmid) | 2ul(50ng) |
Fastpfu DNA polymerase | 1.0μl |
ddH20 | 28.8μl(up to 25ul) |
Temperature program set up
(1×) | 95℃ | 5min | initial denaturation |
(5×) | 95℃ | 20s | denaturation |
1.6μl 52℃ | 20s | annealing | |
72℃ < /td> | 35s | elongation | |
(25×) | 95℃ | 20s | denaturation |
63℃ | 20s e | annealing | |
72℃ | 35s | elongation | |
72℃ | (1×) | 5min | |
(1×) | 4℃ | 10min |
Figure.8
Digestions
Incubated in 37℃ for 90mins
Ligation
Ligation type:1 vector+2 insert fragments
Desired ratio of the amount of substance:
Vector:insert fragment 1:insert fragment 2=1:3:3
Verification of single-clone colonies
PCR verification of Result All chosen colonies are positive, so were the sequencing results.
2013.6.18-7.1
construction of pCDNA3.0-pCMV-RIP1
pCDNA3.0-pCMV-RIP1-1X miR122 complete target
pCDNA3.0-pCMV-RIP1-2X miR122 complete target
2013.7.5-7.31
construction of p199-pEF1a-tTA-PGK-BSD
p199-pEF1a-rtTA-PGK-BSD
p199-pEF1a-tTA advanced-PGK-BSD
p199-pEF1a-RIP1-2XmiR122 targets
p199-pEF1a-RIP3-2XmiR122 targets
p199-pEF1a-eGFP-PGK-BSD
Figure.9
A major problem: DNA Recombination of pEF1a 2013.8.1-8.22
Promoter EF1a we used to drive regulationg parts of tet-on or tet-off system are 537bp in length(supported by professor Lei xiao, Zhejiang University).By NCBI blast search,first 349bp of the 537bp-pEF1a was found to be CPPT,one of the essential elements of inserting backbone plasmid of lentivirus. While the function of CPPT cassette inside pEF1a is yet to figure out, the result of molecular construction suggested that CPPT, which exists both inside 537bp-pEF1a and p199 backbone plasmid, has high ratio of recombination that can interfere the normal inserting process of lentivirus vector and the expression of downstream genes.
Figure 10. The recombination zone
High-ratio recombination between the 2 copies CPPT in plasmid p199-pEF1a(537bp)-gene was first suspected as the forward sequencing primer often present 3 kinds of sequencing results: 1.No signal(most) 2.Double signal(few) 3.single signal(very few)
Figure.11
The recombination mechanism has it proof that the existence of type c plasmid was confirmed by reverse sequencing result (The sequence of some expressed genes driven by pEF1a are short enough to be covered and passed through by reverse sequencing),and that of type B plasmid was confirmed by double sequencing signal. Such recombination mechanism can also be supported by digestion of XhoI restriction site which exists right between CPPT and the 188-bp key region of pEF1a,and in the downstream of expressed genes.
Figure 11. (A).Hypothetical calculation of the result of digestion of XhoI restriction sites.(B).Agarose gel image of the result of digestion of XhoI restriction sites,and the verification of recombination
According to the sequencing and digestion result, the recombination stopped in the downstream of CPPT cassette,unlike a dsDNA whose recombination will terminate at the end of the strain. Due to variable terminating position of recombination on our plasmid p199-537bp pEF1a, the impact on pEF1a and the expression of the genes driven can be different. However,as the first XhoI restriction site appears right between CPPT cassette and 170bps-key region( The 170bps region ,damage of which may has devastating impact on the expression of downstream genes,is found in almost every type of pEF1a), any recombination mode whose terminating point passes through the end of CPPT cassette will destroy XhoI site, therefore the XhoI digestion test(figure.11 B) is acute of any damaging recombination. The variable termination of recombination on our plasmid has its proof that some of plasmids with a single signal of forward sequencing, turned out to be recombined in XhoI digestion test.
Figure 12. Multiple recombination modes
Solutions 2013.8.1-8.22
As the function of CPPT cassette inside pEF1a remains unclear,Several strategies were adopted to deal with the recombination of p199-537bp plasmid and keep such 537bps-pEF1a intact,but none of them has worked out ideally so far.
Strategy A
Reconstruct all p199-537bp pEF1a and transport them into competent bacteria MDS42 strain whose genes expressing recombinases are completed knocked off, instead of Top10 strain.
Result: Failure in collecting single-clone colony, as the competence level of MDS42 strain is extreme low.
Stategy B
Pick up massive single-clone colonies(Top10) and conduct plasmids extraction
Result: All plasmid in XhoI digestion test is positive in recombination.
Then we cutted the growth time in 37℃ thermostat shaker down to 8h in,as we hypothesized that the recombination process mainly occurred during bacteria growth in LB medium. But results were still the same.
Strategy C
Pick up all colonies in the plate and concentrate them for direct plasmids extraction. The plasmids are then retransported to MDS42 strain, whose single-clone colonies will be selected again for plasmids extraction.
Status: currently still underway.
In such case , with the problem of recombination of 537bps-pEF1a unsolved, we chose to replace it with 188bps-pEF1a which was reported still possessing at least 30% of driving intensity of a full-length version pEF1a.And theoretically, the 188bps-pEF1a won’t be recombination capable.
Currently we have successfully constructed p199-188bps pEF1a-tTA-PGK-BSD.
Sun Yat-Sen University, Guangzhou, China
Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China